P53介导的ROS及线粒体通路在Azurin及其联合化疗药物诱导骨肉瘤细胞凋亡的机制探讨
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摘要
背景
骨肉瘤因其恶性程度高、转移早,目前临床上治疗多采用手术配合化疗、放疗的综合疗法,但限于手术治疗的致残率高、假体功能差,放化疗毒副反应大,晚期肿瘤容易形成对化疗药物的耐受,极易复发,再次治疗也极为棘手,故而骨肉瘤的远期生存率较低,疗效难如人意,传统疗法的局限使得骨肉瘤的新型疗法如生物治疗、免疫治疗、基因治疗和微生物纯化产物治疗正日益成为研究和临床的焦点与热点。
近年Yamada研究报道由假单孢细菌分泌的含铜细菌氧化还原蛋白Azurin,可以与P53蛋白结合形成复合体,抑制P53降解,从而稳定P53蛋白。它在体外细胞试验和体内荷瘤裸鼠实验中均可诱导P53野生型的单核巨噬细胞、黑色素瘤细胞、乳腺癌细胞凋亡,而在呈现P53突变或缺失的黑色素瘤细胞、乳腺癌细胞中细胞毒性大大降低,其细胞毒性具有明显的P53选择性。尤为关键的是,在恶性黑色素瘤细胞、乳腺癌细胞的裸鼠体内移植瘤瘤块中,野生型Azurin可以促使移植瘤退变,抑制移植瘤瘤块生长,诱导肿瘤细胞凋亡,而裸鼠体内却没有发现明显的毒副作用。这一发现为活菌产物纯化蛋白Azurin应用于临床肿瘤治疗带来了希望和前景。
浙二骨科研究所杨迪生教授和苗旭东等人已经对Azurin蛋白在人骨肉瘤细胞中开展了一系列体外细胞实验研究。结果如下：Azurin抑制了骨肉瘤的生长,诱导P53野生型U20S细胞凋亡,而对正常肝脏L-02、P53突变型人骨肉瘤MG-63细胞毒性较小。对其分子机制的进一步研究发现：P53蛋白是其发挥凋亡效应的关键因素,Bax的线粒体移位、Bcl-2/Bax比例的下调、caspase-3,8活性增加、线粒体、细胞外Fas受体途径都参与了Azurin诱导U20S细胞凋亡的分子机制。
野生型P53基因是一种抑癌基因,p53的主要功能在于阻滞细胞周期或者诱导细胞凋亡。在p53诱导的细胞凋亡信号途径中有三个关键步骤：(i)氧化还原相关基因的转录诱导；(ii)形成的活性氧(ROS)的形成；(iii)线粒体成分的氧化降解,最终活化caspase导致细胞凋亡。
在骨肉瘤的化疗中,药物毒副反应较大,同时肿瘤由于反复给药极易获得耐药性,复发率较高,因此远期生存率较低。研究表明,肿瘤细胞对细胞凋亡逃避导致的细胞凋亡抵抗(resistance to apoptosis)是肿瘤耐药发生的主要原因之一,细胞内活性氧自由基(ROS)及谷胱甘肽(GSH)还原体系状态水平的改变与细胞凋亡密切相关。因此肿瘤细胞的耐药性与其细胞内的ROS水平差异密切相关。通过调节细胞内GSH水平或升高ROS水平,可以增强化疗药物的凋亡诱导效应。
据此,我们推测Azurin上调p53转录活性,引发胞内氧化还原状态失衡,通过线粒体途径诱导了细胞凋亡,同时,Azurin是否对骨肉瘤化疗药物顺铂(Cisplatin, CDDP)、氨甲喋呤(MTX)、阿霉素(ADM)和异环磷酰胺(IFO)具有化疗增敏效应?并探讨p53、GSH、ROS在Azurin化疗增敏效应中的具体作用机制。
方法
本实验选取三种P53不同基因型的骨肉瘤细胞株(p53野生型U20S、p53突变型MG-63、p53缺失型SAOS-2)以及一种p53野生型正常人外周血单核细胞(PMBCs),应用MTT比色法检测细胞存活率,应用Annexin V、PI双染流式细胞仪检测细胞凋亡率Annexin V positive cell(%),通过流式细胞仪、荧光显微镜检测线粒体膜电位(△Ψm)以及线粒体内膜心磷脂氧化,通过细胞免疫荧光技术、激光共聚焦以及亚细胞器蛋白分离Western blot技术检测线粒体内外膜间蛋白CytochromeC(细胞色素C)、AIF(凋亡诱导因子)释放进入细胞质、细胞质Bax移位线粒体,采用Western blot检测线粒体核心凋亡蛋白Bcl-xL、MCL-1、XIAP、survivin、Bak、 Bid、tBid的表达,通过流式细胞仪检测活性氧(H202)、超氧阴离子(02-)以及一氧化氮(NO),通过酶标仪比色法检测胞内GSH、细胞外液GSH、谷胱甘肽过氧化物酶(GPX)、GSSG(氧化型GSH)、谷胱甘肽还原酶(GR)水平,报告基因质粒pp53-TA-luc和内参Renilla-luc瞬时共转染骨肉瘤细胞SAOS-2(null)、MG-63(mut)、U2OS (wt),荧光检测仪检测上述细胞荧光素酶活性,即p53基因的转录活性。并采用线粒体膜PT孔道抑制剂环孢菌素A(CsA);还原剂Catalase、NAC、 MnTBAP; GSH耗竭剂BSO; P53信号阻断剂PFT-α从正反二个方向验证,以明确线粒体、ROS、P53的具体作用机制。
结果
首先我们观察了Azurin通过P53途径诱导了肿瘤细胞凋亡。
为进一步明确Azurin的P53选择性杀灭肿瘤机理,我们选取了其他组织来源的不同P53基因型背景的肿瘤细胞：携带野生型P53的人肝癌细胞系HepG2细胞株、肺癌细胞株A549、骨肉瘤细胞株U20S；具有P53突变型的肺癌细胞株H322、肝癌细胞株Huh-7细胞、骨肉瘤细胞株MG-63；呈现P53缺失型的肺癌细胞株H1299、肝癌细胞株Hep3B细胞、骨肉瘤细胞株SAOS-2;携带野生型P53的正常人外周血单核细胞(PMBCs)。并通过MTT法观察了Azurin对上述细胞系细胞增殖的影响。
通过MTT试验,我们发现在肺癌、肝癌、骨肉瘤细胞株的不同P53基因背景的肿瘤细胞中,Azurin也同样明显抑制了携带野生型P53的HepG2、A549、U2OS细胞的增殖活性,与之相反,在P53功能性失活的细胞中,Azurin的细胞毒性明显减弱。在上述细胞中,Azurin同样表现出明显的P53选择性细胞毒性效应,同时对正常PMBCs细胞毒性较小。
在p53野生型U20S细胞中,经Azurin处理后,caspase-3活性明显增加,启动Caspase级联反应,PARP发生剪切,诱导细胞凋亡。反之,在p53突变型MG-63、p53缺失型SAOS-2细胞株中,Azurin上调的caspase-3激酶活性、Annexin V positive cell(%)均明显减弱；试验进一步采用P53信号阻断剂PFT-α预孵育U20S细胞,观察其对Azurin作用后的Cleaved-PARP蛋白、激酶caspase-3活性、Annexin V positive cell(%). MTT等指标的影响,我们发现PFT-a几乎完全抑制了Azurin诱导的Caspase-3的活化、PARP的剪切、Annexin V positive (%)的上调；从而证实了Azurin的细胞凋亡分子机理呈现P53依赖性。
其次我们检测了Azurin诱导的线粒体损伤。
P53野生型细胞U20S经Azurin作用后,JC-1红绿荧光比值明显下降,即线粒体膜电位(△Ψm)发生了降低；线粒体NAO荧光强度明显降低,即线粒体内膜心磷脂含量的减少,内膜产生了脂质过氧化损伤,引起线粒体外膜通透性增加；细胞质蛋白Bax单体靶向移位聚集线粒体外膜,构型改变发生寡聚化,构成跨线粒体外膜的蛋白孔道,引发线粒体外膜通透性进一步增加。Bcl-2家族抑凋亡蛋白Bcl-xL和MCL-1、XIAP的蛋白表达水平自8h时间点开始降低,促凋亡蛋白Bak的表达量呈现时间依赖性上调,同时截短型Bid(tBid)从4h时间点开始出现,24h时间点tBid蛋白表达量达到峰值,Bid蛋白发生了剪切。与心磷脂结合的细胞色素c得以解偶联,线粒体内外膜之间的蛋白Cytochrome C(细胞色素C)、AIF(凋亡诱导因子)经蛋白孔道大量释放进入细胞质,启动caspase的级联反应,激活下游效应器caspase-3,AIF进而易位细胞核,在核内通过和DNA结合导致染色质聚集并断裂。
值得注意的是,在p53突变型MG-63、p53缺失型SAOS-2细胞以及正常人外周血单核细胞(PMBCs)中,经Azurin处理后,线粒体膜电位△Ψm、线粒体内膜心磷脂含量均未见明显改变,提示P53信号途径与Azurin诱导的线粒体损伤有关。
为了明确线粒体途径在Azurin诱导细胞凋亡中的作用,我们采用线粒体膜PT孔道抑制剂环孢菌素A(CsA)预先孵育U20S细胞,CsA明显抑制了Azurin增强的caspase-3活性、Cleaved-PARP蛋白的上调以及细胞凋亡。但却对Azurin诱导的胞内的ROS水平上调、GSH水平降低近乎没有影响,这提示线粒体通道开放事件发生较晚,是位于氧化还原系统失衡发生的下游事件。
再次,我们观察了Azurin诱导的细胞内氧化还原状态失衡。
Azurin在P53野生型U20S细胞中,迅速引发DCF(H2O2), ethidium (O2-)荧光强度的显著上调,却对DAF-FM (NO)荧光强度没有影响,即Azurin主要引起了过氧化氢(H202)、超氧阴离子(02-)这二种自由基的上升,却对一氧化氮(NO)没有影响。
同时我们观察了Azurin对U20S细胞GSH还原系统的影响,我们发现Azurin抑制谷胱甘肽过氧化物酶(GPx)的合成与酶的活性,氧化型GSSG含量大大增加,降低了GSH还原系统的抗氧化能力,诱发GSH释放进入细胞外液,进一步耗竭细胞内GSH,促发氧化还原系统失衡。
应用GSH的前体抗氧化剂NAC预处理P53野生型U20S细胞,我们发现NAC明显抑制了Azurin诱导的胞内GSH水平降低以及ROS上调,也阻止了Azurin促发的线粒体膜电位的下降以及内膜心磷脂的过氧化,并最终抑制了caspase活化介导的细胞凋亡。从而提示Azurin引发的胞内氧化还原状态失衡是介于线粒体损伤发生之前的上游事件。
试验进一步采用还原剂过氧化氢(H202)酶Catalase预处理P53野生型细胞U2OS, Catalase抑制了Azurin诱导的胞内ROS水平上调以及细胞凋亡,却对Azurin所致的GSH水平降低没有影响,这表明Azurin在U20S细胞中首先引发了GSH水平的下降,进而导致了氧自由基过氧化氢(H2O2)的积聚。GSH下降是先于ROS积聚发生的上游事件。
我们还采用超氧阴离子清除剂MnTBAP预处理P53野生型细胞U20S，发现其对Azurin诱导的胞内氧化应激、caspase活性均没有影响,这表明虽然超氧阴离子水平呈现上调,但其并未参与Azurin诱导的细胞凋亡。
同U20S细胞相比,P53突变型细胞MG63、p53缺失型SAOS-2经Azurin处理后,过氧化氢(H2O2)、超氧阴离子(O2-)、细胞外液GSH、细胞内GSH含量、谷胱甘肽过氧化物酶(GPX)活性均未见明显改变。
BSO抑制细胞内GSH合成,P53突变型MG63、p53缺失型SAOS-2细胞经BSO作用后,胞内GSH显著下降,氧自由基水平急剧增高,线粒体△Ψm明显降低,线粒体内膜脂质发生过氧化,Caspase活化,诱导细胞发生凋亡。BSO模拟了Azurin在U20S中的细胞毒性效应。
上述结果表明GSH耗竭、ROS的积聚在Azurin诱导的细胞凋亡中发挥着重要作用,并且氧化应激有可能受p53途径调控。
最后,我们观察了P53信号通路、氧化应激、线粒体损伤、细胞凋亡间的相互关系。
我们将报告基因质粒pp53-TA-luc和内参Renilla-luc瞬时共转染肺癌细胞株H1299(null). H322(mut)、A549(wt);肝癌细胞株Hep3B (null). Huh-7(mut)、 HepG2(wt);骨肉瘤细胞株SAOS-2(null)、MG-63(mut)、U20S (wt),荧光检测仪检测荧光素酶活性,我们发现Azurin增强了p53基因的转录活性。
试验进一步采用p53抑制剂PFT-α(pifithrinalpha,PFT-α)干扰试验,阻断U2OS的P53信号途径,结果显示PFT-α的预处理抑制了Azurin触发的GPX活性降低、GSH下降、ROS上调；线粒体膜电位△Ψm降低、内膜心磷脂氧化；Bax移位线粒体；细胞色素C、AIF释放进入胞质。这表明Azurin介导的氧化还原状态失衡、线粒体损伤受p53信号途径调控。
此外,实验结果还显示：在U2OS细胞中,NAC预处理抑制了Azurin上调的P53转录活性,而GSH耗竭剂BSO则在U2OS细胞中模拟了Azurin上调的P53转录活性,从而提示氧化还原状态的失衡正反馈激活了P53信号途径。
上述研究结果表明,P53信号通路及其调控的GSH代谢、ROS水平、线粒体途径,在Azurin诱导的靶细胞凋亡过程中扮演着重要角色。
我们选取骨肉瘤化疗常用药物如顺铂(Cisplatin, CDDP).氨甲喋呤(MTX)、阿霉素(ADM)和异环磷酰胺(IFO),在U20S、MG-63、SAOS-2细胞中,进行Azurin的药物增敏活性筛选,以验证小剂量Azurin联合低浓度化疗药物是否对骨肉瘤细胞的杀伤具有增敏效应?并就P53、ROS、GSH在其中的分子机制进一步探讨。
在顺铂、氨甲喋呤的药敏试验中,在U2OS细胞中,MTT显示,低剂量Azurin显著增加顺铂、氨甲喋呤的细胞毒性,联合给药具有协同效应。同时,也引发了GSH下降、ROS增加。还原剂NAC p53抑制剂PFT-α(pifithrinalpha,PFT-a)预处理U20S后,明显抑制了联合给药组所致的GSH降低、ROS上调以及细胞存活率的下降。而在P53功能缺失的其余二株MG-63、SAOS-2细胞中,联合给药组缺乏协同效应,同时,MG-63、SAOS-2胞内GSH、ROS水平也几乎没有改变。
以上数据表明了在Azurin联合顺铂、氨甲喋呤组的增敏效应中,P53及其调控的氧化还原状态失衡尤为关键。
在阿霉素、异环磷酰胺的药敏试验中,有趣的是,在三种MG-63、SAOS-2、 U2OS细胞中,小剂量Azurin都显著增强了阿霉素、异环磷酰胺的细胞毒性,在联合给药中,我们没有观察到GSH下降、ROS增加,还原剂NAC、p53抑制剂PFT-α(pifithrinalpha,PFT-α)的预处理对联合给药组所致的细胞存活率的下降也近乎没有影响,从而提示在MG-63、SAOS-2、U2OS中,Azurin对阿霉素、异环磷酰胺细胞毒性的增敏效应与P53、ROS途径无关。这表明在P53功能性缺失的肿瘤细胞中,Azurin也可对化疗药物的细胞毒性具有协同增敏效应,从而避免其局限于P53野生型肿瘤细胞。
此外,试验还发现对于人外周血单核细胞(PBMC)细胞,Azurin联合顺铂、氨甲喋呤、阿霉素、异环磷酰胺给药组的细胞毒性相对较小。这意味着,联合给药组既避免了单独给药时肿瘤细胞的获得性耐药,又使得其对正常体细胞的毒副反应大大降低。
结论
综上所述,在杨迪生教授、苗旭东等人的工作基础上,我们进一步探讨了P53、 ROS、线粒体途径在Azurin细胞毒性中的分子机制：Azurin增强p53转录活性↑→p53胞内水平↑→谷胱甘肽过氧化物酶(GPX)↓→GSH↓→ROS↑→线粒体内膜心磷脂氧化→线粒体膜电位(△Ψm)↓→Bax移位线粒体→细胞色素C、AIF释放进入胞质→激活线粒体途径→caspase活化→骨肉瘤细胞凋亡。P53基因调控的氧化还原状态失衡(GSH耗竭和ROS增高)在其中尤为关键。此外,通过P53依赖性或者非P53依赖性途径,Azurin与低剂量化疗药物如顺铂(Cisplatin, CDDP)、氨甲喋呤(MTX)、阿霉素(ADM)和异环磷酰胺(IFO)的联合给药具有协同效应,Azurin对上述化疗药物体外骨肉瘤细胞的细胞毒性具有增敏效应,其P53依赖性增敏机制与P53调控的氧化应激密切相关,而其不依赖于P53的增敏机制则与氧自由基水平无关。
本论文的研究结果将不仅有助于发现Azurin的可能作用靶点以及信号通路,还为Azurin的临床前期开发应用提供充分的实验依据,也为今后Azurin应用于临床骨肉瘤化疗的联合用药提供了安全有效的分子策略。
Backgroud:
Osteosarcoma because of its high degree of malignancy, early metastasis, clinical treatment with surgery combined with chemotherapy and radiotherapy treatments, but limited to the surgical treatment of high morbidity and poor function of the prosthesis, put the side effects of chemotherapy, advanced tumors easy to form a tolerance to chemotherapeutic drugs, vulnerable to relapse, re-treatment is also extremely difficult, and therefore lower long-term survival of osteosarcoma difficult to satisfactory efficacy, the limitations of traditional therapy makes osteosarcoma new therapies such as biological therapy, immunotherapy therapy, gene therapy, and microbial treatment of the purified product are increasingly becoming the focus of research and clinical and hotspots.
Yamada reported in recent years by Pseudomonas bacteria secrete copper-containing bacterial redox protein Azurin, can combine to form complexes with the P53protein, inhibition of P53degradation, thereby stabilizing the P53protein.It can be induced in vitro cell assays and in vivo tumor-bearing nude mice P53wild type mononuclear macrophages, melanoma cells, breast cancer cells, while P53mutation or deletion of melanoma cells in the rendered breast cancer cells, the cytotoxicity is greatly reduced, its cytotoxic obvious p53selectivity.Particularly critical in malignant melanoma cells, breast cancer cells in nude mice transplanted tumor, tumor size, and the wild-type Azurin can promote the degeneration of the transplanted tumors, inhibiting the growth of transplanted tumors tumor block induction of tumor cell apoptosis, while nude mice The body was found no significant side effects.This finding viable cells product purified protein the Azurin used in clinical tumor therapy to bring hope and prospects.
Zhejiang Second Orthopaedic Institute Professor Yang Disheng and Miao Xudong et al Azurin protein in human osteosarcoma cells to carry out a series of in vitro experimental study.The results are as follows:Azurin inhibited the growth of osteosarcoma induced P53wild type U2OS cell apoptosis, while normal liver L-02the P53mutant human osteosarcoma MG-63cells less toxic.Further study its molecular mechanisms:P53protein is play the key factors of the apoptotic effects the Bax mitochondrial shift of Bcl-2/Bax ratio downward, caspase-3,8increased activity, mitochondrial, extracellular Fas by pathway Azurin-induced the U2OS cell apoptosis molecular mechanisms are involved.
Tumor cells escape apoptosis caused apoptosis resistance (resistance to apoptosis) is one of the main reasons of tumor drug resistance, restore the system state level of intracellular reactive oxygen species (ROS) and glutathione (GSH) change is closely related to the apoptosis.
The wild-type p53gene is a tumor suppressor gene, the main function of p53is to arrest the cell cycle or induce apoptosis.There are three key steps in the p53-induced apoptosis signaling pathways:(i) oxidation reduction related gene transcription induced;(ii) the formation of reactive oxygen species (ROS) is formed; component (iii) mitochondrial oxidative degradation, and ultimately activation caspase leading to apoptosis.
In osteosarcoma chemotherapy drug toxicity reaction, while tumor recurrence rate was high due to repeated administration can easily acquire resistance, and therefore the long-term survival rate is lower.Studies have shown that the the ROS level differences related to the resistance of tumor cells to their cells.Through the regulation of intracellular GSH levels or elevated ROS levels can enhance the apoptosis-inducing effects of chemotherapy drugs.
Accordingly, we speculate Azurin raised p53transcriptional activity, triggered intracellular oxidation reduction state of imbalance through the mitochondrial pathway induction of apoptosis, and at the same time, Azurin of osteosarcoma tumor chemotherapy drug cis-platinum (Cisplatin, of CDDP), methotrexate chatter methotrexate (MTX). adriamycin (ADM) and ifosfamide (IFO) with chemotherapy sensitizing effect?And to explore the the p53, GSH, ROS in Azurin chemosensitizing effect of the specific mechanism of action.
Methods:
In our study, three different genotypes of P53osteosarcoma cell line (p53wild-type U2OS, p53mutant MG-63, p53-deficient SAOS-2) and a p53wild-type normal Human peripheral blood mononuclear cells (PMBCs), by MTT colorimetric assay of cell viability, application of Annexin V and PI double staining flow cytometry apoptosis rate Annexin V positive cell (%) by flow cytometry, fluorescence microscopy detection of mitochondrial membrane potential (ΔΨm) and mitochondrial inner membrane heart phospholipids oxidation by immunofluorescence techniques, laser confocal subcellular protein separation Cytochrome C (cytochrome C) Western blot analysis of mitochondrial outer membrane protein, AIF (apoptosis-inducing factor) release into the cytoplasm, cytoplasmic Bax shift mitochondria, Western blot analysis of mitochondrial core apoptotic protein Bcl-xL of MCL-1, XIAP, survivin, Bak, Bid, tBid of expression by flow cytometry reactive oxygen species (H202), superoxide anion (O2-) and nitric oxide (NO), through the the microplate colorimetric detection intracellular GSH, extracellular fluid GSH, glutathione peroxidase (GPX), GSSG (oxidized form of GSH), glutathione reductase (GR) level, pp53-TA-luc reporter plasmid and the internal reference Renilla-luc transiently co-transfected osteosarcoma cells SAOS-2(null), MG-63(mut), U2OS (wt), the fluorescence detector detects the above cell luciferase activity, namely the transcriptional activity of the p53gene.And mitochondrial membrane PT pore inhibitor cyclosporine A (CsA); reductant Catalase, NAC, MnTBAP; GSH depleting agent BSO; P53signal blocking agent PFT-a verified from two positive and negative direction to clear mitochondria ROS, P53specific mechanism of action.
Result:
First, we observed Azurin P53pathway induced tumor cell apoptosis.
Further clear of Azurin the P53selectively kill tumor mechanism, we have selected different P53genotype background of tumor cells in other tissue sources:53people carrying wild-type P hepatoma cell line HepG2cell lines, lung cancer cell line A549, flesh and blood tumor cell line U2OS; P53mutant lung cancer cell line H322, hepatoma cell line Huh-7cells, osteosarcoma cell line MG-63; render P53deletion lung cancer cell lines HI299hepatoma cell line Hep3B cells, osteosarcoma SAOS-2cell lines; carrying the wild-type P53normal human peripheral blood the monocytes (PMBCs).Azurin proliferation of these cell lines by MTT assay.
By MTT assay, we found that the tumor cells in the background of P53gene in lung cancer, liver cancer, osteosarcoma cell lines, Azurin also significantly inhibited with wild-type P53the HepG2, A549, U2OS cells proliferation activity, whom In contrast, in the cells of P53functional inactivation AZURIN cell toxicity is significantly weakened.In these cells, Azurin perform equally obvious P53selective cytotoxic effect, simultaneously less toxic of normal PMBCs cells.
Azurin treatment in p53wild-type U2OS cells, caspase-3activity was significantly increased, start caspase cascade, PARP occurred shear induced apoptosis.Conversely, p53mutated MG-63, p53-deficient SAOS-2cell lines Azurin upregulation of caspase-3kinase activity, Annexin V positive cell (%) were significantly weakened; trials further a P53signal blocker PFT-a preincubation U2OS cells, were observed on the the Azurin role Cleaved-PARP protein kinase caspase-3activity, Annexin V positive cell (%), MTT indicators, we find that PFT-a almost completely inhibited Azurin induced Caspase-3activation, PARP a shear, Annexin V positive (%) of the raised; thus confirming the molecular mechanism of Azurin apoptosis presented P53-dependent manner.
Secondly, we detect the the Azurin induced mitochondrial damage.
P53wild-type cells U2OS after Azurin role, JC-1red and green fluorescence ratio decreased mitochondrial membrane potential (ΔΨm) happened; mitochondrial NAO fluorescence intensity was significantly reduced, and reduce mitochondrial endometrial cardiolipin content endometrial produce a shift gathered mitochondrial outer membrane lipid peroxidation, causing increased permeability of the outer mitochondrial membrane; cytoplasmic protein Bax monomer targeted protein conformational change occurred oligomerization to form across the mitochondrial outer membrane pore, lead to mitochondrial further increase in membrane permeability.Bcl-2family inhibiting apoptotic protein Bcl-xL and MCL-1, XIAP protein expression levels of self-8h time point reduced to promote the expression of apoptotic protein Bak presents a time-dependent increase, while the truncated Bid (tBid) from the4h time to point began to appear, the24h time point tBid the protein expression levels peak of Bid Protein shear.Cytochrome C combined with cardiolipin be uncoupling between the inner and outer mitochondrial membrane protein Cytochrome C (Cytochrome C), AIF (apoptosis inducing factor) protein pore massive release into the cytoplasm, start caspase cascade activation of downstream effector caspase-3, AIF thus translocation nucleus and DNA binding in the nuclear cause staining quality gathering and fracture.
It is noteworthy that in p53mutant MG-63, p53-deficient SAOS-2cells and normal human peripheral blood mononuclear cells (PMBCs) Azurin treatment, mitochondrial membrane potential ΔΨm, mitochondrial inner membrane center phospholipid content did not change significantly, suggesting that p53signaling pathways Azurin-induced mitochondrial damage.
To clear the mitochondrial pathway Azurin induced apoptosis, mitochondrial membrane PT pore inhibitor cyclosporine A (CsA) pre-incubated U2OS cells, CsA significantly inhibited the the Azurin enhanced caspase-3activity, Cleaved-PARP The upregulation of the protein, and apoptosis.But raised on Azurin induced intracellular ROS levels, GSH levels decreased almost no effect, suggesting that mitochondrial channel opening events later, in the redox system imbalance occurs downstream events.
Again, we observed Azurin-induced cell redox state of imbalance.
Azurin in a P53wild-type U2OS cells, quickly lead to the DCF (H2O2), ethidium (O2-) significant upregulation of the fluorescence intensity, but then the fluorescence intensity of the DAF-FM (NO) does not affect, i.e. The Azurin mainly caused too hydrogen peroxide (H2O2) and superoxide anion (O2-) of these two kinds of radicals rise, but then the nitric oxide (NO) no effect.
Meanwhile, we observed the Azurin U20S cells GSH reduction system, we found Azurin suppression Synthesis and enzyme activity of glutathione peroxidase (GPX), oxidized GSSG content increased greatly reduces the antioxidant capacity of the GSH reduction system, to induce GSH release into the extracellular fluid, further depletion of intracellular GSH, promote hair redox system imbalance.
Application of GSH precursor antioxidants NAC pretreatment p53wild-type U20S cells, we found that NAC significantly inhibited the Azurin induced intracellular GSH level reduced and ROS upward, also prevent a decline in mitochondrial membrane potential and intimal Azurin priming heart phospholipid peroxidation, and eventually suppressed caspase activation mediated apoptosis.Which prompted Azurin triggered intracellular redox state imbalance between mitochondrial damage occurs before the upstream events.
Test further reducing agent, hydrogen peroxide (H2O2) the enzyme Catalase pretreatment P53wild-type cells U20S Catalase suppression Azurin induced intracellular ROS levels increase, and apoptosis, but then the Azurin due to the reduced GSH levels did not impact, which indicates that the decline in the level of GSH Azurin first initiator in U2OS cells, leading to oxygen radicals of hydrogen peroxide (H2O2) accumulation.GSH decreased upstream events prior to ROS accumulation occurred.
We also used the superoxide anion scavenger MnTBAP pretreatment P53wild type cells U2OS the the Azurin induced intracellular oxidative stress, caspase activity had no effect, suggesting that superoxide anion levels presented raised, but did not participate in The Azurin induced apoptosis.
Compared with U2OS cells, P53mutant cells of MG63of p53-deficient SAOS-2after Azurin processing, hydrogen peroxide (H2O2), superoxide anion (O2-) cell extracellular fluid GSH intracellular GSH content, glutathione Gan peptide peroxidase (GPX) activity showed no significant change.
The BSO inhibition of intracellular GSH synthesis P53mutant of MG63and p53-deficient SAOS-2cells after BSO role of intracellular GSH was significantly decreased, and a sharp increase in the level of oxygen free radicals, significantly decreased the ΔΨm, mitochondrial inner membrane lipid peroxidation occurred, Caspase activation and induce apoptosis.The BSO analog Azurin in U2OS cytotoxic effect.
The above results indicate that GSH depletion, accumulation of ROS-induced apoptosis in Azurin plays an important role, and oxidative stress may be affected by the p53pathway regulation.
Finally, we observed p53signaling pathway, the relationship between oxidative stress, mitochondrial damage, apoptosis.
We will pp53-TA-luc reporter plasmid and the internal reference Renilla-luc instantaneous co-transfected lung cancer cell lines H1299(null), H322(mut), A549(wt); hepatoma cell line Hep3B (null), Huh-7(mut was), HepG2(wt); osteosarcoma cell line, SAOS-2(null), MG-63(MUT), U2OS,(by weight), the fluorescent detector detecting the luciferase activity, we found to The Azurin enhance the transcriptional activity of the p53gene.
Further test p53inhibitor PFT-α (pifithrinalpha, PFT-α) interference test, blocked U2OS P53signaling pathways, and results showed that PFT-a pretreatment inhibits Azurin triggered GPX activity decreased, GSH decreased, ROS raised; mitochondrial membrane potential ΔΨm lower endometrial cardiolipin oxidation; the Bax shift mitochondria; cytochrome C, AIF release into the cytoplasm.This indicates that the redox state of imbalance Azurin mediated mitochondrial damage by the p53signaling pathway regulation.
In addition, the results also show that:U2OS cells, NAC pretreatment inhibited Azurin upregulation of p53transcriptional activity, GSH depletion agent BSO in U2OS cells to simulate the the Azurin raised p53transcriptional activity, suggesting the redox state of imbalance Positive feedback activation of the p53signaling pathway.
The results of the study show that the P53signaling pathway and its regulation of GSH metabolism, ROS levels, mitochondrial pathway in the Azurin induced target cell apoptosis process plays an important role.
We selected the commonly used drugs such as osteosarcoma chemotherapy Cisplatin (Cisplatin, CDDP), methotrexate (MTX), adriamycin (ADM) and Ifosfamide (IFO), U2OS MG-63, SAOS-2cells, Azurin drug-sensitizing activity screening to verify that small doses Azurin combined with low concentrations of chemotherapy drugs osteosarcoma cells destruction sensitizing effect?And P53, ROS, GSH in which to further explore the molecular mechanisms.
Susceptibility testing of cisplatin, methotrexate, U2OS cells, MTT showed that low-dose the Azurin significant increase cisplatin, methotrexate cytotoxicity, administered in combination have a synergistic effect.At the same time, it also raised GSH decline, ROS increase.Reducing agent NAC, p53inhibitor PFT-a (pifithrinalpha, PFT-a) after pretreatment U2OS significantly inhibited due to the co-administered group of GSH decreased ROS raised as well as the reduction in cellular viability.In P53loss of function of the remaining two MG-63, SAOS-2cells, combination group lack of synergy effects, the MG-63, SAOS-2intracellular GSH of ROS levels are almost no change.
These data suggest that the P53and its regulation of the redox state of imbalance is particularly critical in Azurin combination with cisplatin, methotrexate group sensitizing effect.
Doxorubicin, ifosfamide susceptibility testing, it is interesting that three kinds of MG-63, SAOS-2, U2OS cells, small doses of Azurin significantly enhanced doxorubicin, ifosfamide cytotoxicity, co-administration, we have not observed GSH decline, ROS increases, reducing agent NAC p53inhibitor PFT-a (pifithrinalpha, PFT-a) pretreatment cell viability due to the co-administered group decline in almost no effect, which prompted the MG-63, SAOS-2, U2OS, Azurin doxorubicin, different sensitizing effect of cyclophosphamide cytotoxicity P53, ROS pathway unrelated.This shows that in the p53functional deletion of tumor cells, Azurin also be synergistic sensitizing effect of cytotoxic chemotherapy drugs, thus avoiding its limited to P53wild type tumor cells.
In addition, the tests also found that the cells of the human peripheral blood mononuclear cells (PBMC), AZURIN cisplatin, methotrexate, adriamycin, ifosfamide administration group cytotoxic relatively small.This means that the co-administration group only avoids acquired drug resistance of tumor cells when administered alone, but also makes its toxic side effects on normal cells greatly reduced.
Conclusion:
In In summary, on the basis of the work of Professor Yang Disheng, Miao Xudong et al, we further investigate the P53, ROS, mitochondrial pathway of the molecular mechanisms of cytotoxicity in Azurin:Azurin enhanced p53transcriptional activity↑→p53intracellular levels→GSH↓of ROS, the↑→glutathione peroxidase (GPX)↓↑→mitochondrial oxidation of endometrial cardiolipin→mitochondrial membrane potential (ΔΨ m)↓→Bax shift mitochondrial→cytochrome C, AIF release into cell the quality→activation of the mitochondrial pathway→caspase activation→apoptosis of osteosarcoma.Redox state of the P53gene regulatory imbalance (GSH depletion and increased ROS) in which is particularly critical.In addition, the P53-dependent or non-dependent pathway of P53Azurin with low doses of chemotherapy drugs such as cisplatin (Cisplatin, CDDP), methotrexate methotrexate (MTX), adriamycin (ADM) and ifosfamide (IFO) to the United The drugs have a synergistic effect, Azurin sensitizing effect of the chemotherapy drugs osteosarcoma cells in vitro cytotoxic, oxidative stress is closely related to its P53-dependent the sensitizing mechanism with P53control, which does not depend on the P53sensitizing mechanism has nothing to do with the level of oxygen free radicals.
The findings of this paper will not only help to identify the Azurin the possible targets of signaling pathways also provide sufficient experimental evidence of Azurin the preclinical development and application, and also for the future Azurin used in clinical osteosarcoma chemotherapy combination provides a safe and effective molecular strategies.
骨肉瘤因其恶性程度高、转移早,目前临床上治疗多采用手术配合化疗、放疗的综合疗法,但限于手术治疗的致残率高、假体功能差,放化疗毒副反应大,晚期肿瘤容易形成对化疗药物的耐受,极易复发,再次治疗也极为棘手,故而骨肉瘤的远期生存率较低,疗效难如人意,传统疗法的局限使得骨肉瘤的新型疗法如生物治疗、免疫治疗、基因治疗和微生物纯化产物治疗正日益成为研究和临床的焦点与热点。
近年Yamada研究报道由假单孢细菌分泌的含铜细菌氧化还原蛋白Azurin,可以与P53蛋白结合形成复合体,抑制P53降解,从而稳定P53蛋白。它在体外细胞试验和体内荷瘤裸鼠实验中均可诱导P53野生型的单核巨噬细胞、黑色素瘤细胞、乳腺癌细胞凋亡,而在呈现P53突变或缺失的黑色素瘤细胞、乳腺癌细胞中细胞毒性大大降低,其细胞毒性具有明显的P53选择性。尤为关键的是,在恶性黑色素瘤细胞、乳腺癌细胞的裸鼠体内移植瘤瘤块中,野生型Azurin可以促使移植瘤退变,抑制移植瘤瘤块生长,诱导肿瘤细胞凋亡,而裸鼠体内却没有发现明显的毒副作用。这一发现为活菌产物纯化蛋白Azurin应用于临床肿瘤治疗带来了希望和前景。
浙二骨科研究所杨迪生教授和苗旭东等人已经对Azurin蛋白在人骨肉瘤细胞中开展了一系列体外细胞实验研究。结果如下：Azurin抑制了骨肉瘤的生长,诱导P53野生型U20S细胞凋亡,而对正常肝脏L-02、P53突变型人骨肉瘤MG-63细胞毒性较小。对其分子机制的进一步研究发现：P53蛋白是其发挥凋亡效应的关键因素,Bax的线粒体移位、Bcl-2/Bax比例的下调、caspase-3,8活性增加、线粒体、细胞外Fas受体途径都参与了Azurin诱导U20S细胞凋亡的分子机制。
野生型P53基因是一种抑癌基因,p53的主要功能在于阻滞细胞周期或者诱导细胞凋亡。在p53诱导的细胞凋亡信号途径中有三个关键步骤：(i)氧化还原相关基因的转录诱导；(ii)形成的活性氧(ROS)的形成；(iii)线粒体成分的氧化降解,最终活化caspase导致细胞凋亡。
在骨肉瘤的化疗中,药物毒副反应较大,同时肿瘤由于反复给药极易获得耐药性,复发率较高,因此远期生存率较低。研究表明,肿瘤细胞对细胞凋亡逃避导致的细胞凋亡抵抗(resistance to apoptosis)是肿瘤耐药发生的主要原因之一,细胞内活性氧自由基(ROS)及谷胱甘肽(GSH)还原体系状态水平的改变与细胞凋亡密切相关。因此肿瘤细胞的耐药性与其细胞内的ROS水平差异密切相关。通过调节细胞内GSH水平或升高ROS水平,可以增强化疗药物的凋亡诱导效应。
据此,我们推测Azurin上调p53转录活性,引发胞内氧化还原状态失衡,通过线粒体途径诱导了细胞凋亡,同时,Azurin是否对骨肉瘤化疗药物顺铂(Cisplatin, CDDP)、氨甲喋呤(MTX)、阿霉素(ADM)和异环磷酰胺(IFO)具有化疗增敏效应?并探讨p53、GSH、ROS在Azurin化疗增敏效应中的具体作用机制。
方法
本实验选取三种P53不同基因型的骨肉瘤细胞株(p53野生型U20S、p53突变型MG-63、p53缺失型SAOS-2)以及一种p53野生型正常人外周血单核细胞(PMBCs),应用MTT比色法检测细胞存活率,应用Annexin V、PI双染流式细胞仪检测细胞凋亡率Annexin V positive cell(%),通过流式细胞仪、荧光显微镜检测线粒体膜电位(△Ψm)以及线粒体内膜心磷脂氧化,通过细胞免疫荧光技术、激光共聚焦以及亚细胞器蛋白分离Western blot技术检测线粒体内外膜间蛋白CytochromeC(细胞色素C)、AIF(凋亡诱导因子)释放进入细胞质、细胞质Bax移位线粒体,采用Western blot检测线粒体核心凋亡蛋白Bcl-xL、MCL-1、XIAP、survivin、Bak、 Bid、tBid的表达,通过流式细胞仪检测活性氧(H202)、超氧阴离子(02-)以及一氧化氮(NO),通过酶标仪比色法检测胞内GSH、细胞外液GSH、谷胱甘肽过氧化物酶(GPX)、GSSG(氧化型GSH)、谷胱甘肽还原酶(GR)水平,报告基因质粒pp53-TA-luc和内参Renilla-luc瞬时共转染骨肉瘤细胞SAOS-2(null)、MG-63(mut)、U2OS (wt),荧光检测仪检测上述细胞荧光素酶活性,即p53基因的转录活性。并采用线粒体膜PT孔道抑制剂环孢菌素A(CsA);还原剂Catalase、NAC、 MnTBAP; GSH耗竭剂BSO; P53信号阻断剂PFT-α从正反二个方向验证,以明确线粒体、ROS、P53的具体作用机制。
结果
首先我们观察了Azurin通过P53途径诱导了肿瘤细胞凋亡。
为进一步明确Azurin的P53选择性杀灭肿瘤机理,我们选取了其他组织来源的不同P53基因型背景的肿瘤细胞：携带野生型P53的人肝癌细胞系HepG2细胞株、肺癌细胞株A549、骨肉瘤细胞株U20S；具有P53突变型的肺癌细胞株H322、肝癌细胞株Huh-7细胞、骨肉瘤细胞株MG-63；呈现P53缺失型的肺癌细胞株H1299、肝癌细胞株Hep3B细胞、骨肉瘤细胞株SAOS-2;携带野生型P53的正常人外周血单核细胞(PMBCs)。并通过MTT法观察了Azurin对上述细胞系细胞增殖的影响。
通过MTT试验,我们发现在肺癌、肝癌、骨肉瘤细胞株的不同P53基因背景的肿瘤细胞中,Azurin也同样明显抑制了携带野生型P53的HepG2、A549、U2OS细胞的增殖活性,与之相反,在P53功能性失活的细胞中,Azurin的细胞毒性明显减弱。在上述细胞中,Azurin同样表现出明显的P53选择性细胞毒性效应,同时对正常PMBCs细胞毒性较小。
在p53野生型U20S细胞中,经Azurin处理后,caspase-3活性明显增加,启动Caspase级联反应,PARP发生剪切,诱导细胞凋亡。反之,在p53突变型MG-63、p53缺失型SAOS-2细胞株中,Azurin上调的caspase-3激酶活性、Annexin V positive cell(%)均明显减弱；试验进一步采用P53信号阻断剂PFT-α预孵育U20S细胞,观察其对Azurin作用后的Cleaved-PARP蛋白、激酶caspase-3活性、Annexin V positive cell(%). MTT等指标的影响,我们发现PFT-a几乎完全抑制了Azurin诱导的Caspase-3的活化、PARP的剪切、Annexin V positive (%)的上调；从而证实了Azurin的细胞凋亡分子机理呈现P53依赖性。
其次我们检测了Azurin诱导的线粒体损伤。
P53野生型细胞U20S经Azurin作用后,JC-1红绿荧光比值明显下降,即线粒体膜电位(△Ψm)发生了降低；线粒体NAO荧光强度明显降低,即线粒体内膜心磷脂含量的减少,内膜产生了脂质过氧化损伤,引起线粒体外膜通透性增加；细胞质蛋白Bax单体靶向移位聚集线粒体外膜,构型改变发生寡聚化,构成跨线粒体外膜的蛋白孔道,引发线粒体外膜通透性进一步增加。Bcl-2家族抑凋亡蛋白Bcl-xL和MCL-1、XIAP的蛋白表达水平自8h时间点开始降低,促凋亡蛋白Bak的表达量呈现时间依赖性上调,同时截短型Bid(tBid)从4h时间点开始出现,24h时间点tBid蛋白表达量达到峰值,Bid蛋白发生了剪切。与心磷脂结合的细胞色素c得以解偶联,线粒体内外膜之间的蛋白Cytochrome C(细胞色素C)、AIF(凋亡诱导因子)经蛋白孔道大量释放进入细胞质,启动caspase的级联反应,激活下游效应器caspase-3,AIF进而易位细胞核,在核内通过和DNA结合导致染色质聚集并断裂。
值得注意的是,在p53突变型MG-63、p53缺失型SAOS-2细胞以及正常人外周血单核细胞(PMBCs)中,经Azurin处理后,线粒体膜电位△Ψm、线粒体内膜心磷脂含量均未见明显改变,提示P53信号途径与Azurin诱导的线粒体损伤有关。
为了明确线粒体途径在Azurin诱导细胞凋亡中的作用,我们采用线粒体膜PT孔道抑制剂环孢菌素A(CsA)预先孵育U20S细胞,CsA明显抑制了Azurin增强的caspase-3活性、Cleaved-PARP蛋白的上调以及细胞凋亡。但却对Azurin诱导的胞内的ROS水平上调、GSH水平降低近乎没有影响,这提示线粒体通道开放事件发生较晚,是位于氧化还原系统失衡发生的下游事件。
再次,我们观察了Azurin诱导的细胞内氧化还原状态失衡。
Azurin在P53野生型U20S细胞中,迅速引发DCF(H2O2), ethidium (O2-)荧光强度的显著上调,却对DAF-FM (NO)荧光强度没有影响,即Azurin主要引起了过氧化氢(H202)、超氧阴离子(02-)这二种自由基的上升,却对一氧化氮(NO)没有影响。
同时我们观察了Azurin对U20S细胞GSH还原系统的影响,我们发现Azurin抑制谷胱甘肽过氧化物酶(GPx)的合成与酶的活性,氧化型GSSG含量大大增加,降低了GSH还原系统的抗氧化能力,诱发GSH释放进入细胞外液,进一步耗竭细胞内GSH,促发氧化还原系统失衡。
应用GSH的前体抗氧化剂NAC预处理P53野生型U20S细胞,我们发现NAC明显抑制了Azurin诱导的胞内GSH水平降低以及ROS上调,也阻止了Azurin促发的线粒体膜电位的下降以及内膜心磷脂的过氧化,并最终抑制了caspase活化介导的细胞凋亡。从而提示Azurin引发的胞内氧化还原状态失衡是介于线粒体损伤发生之前的上游事件。
试验进一步采用还原剂过氧化氢(H202)酶Catalase预处理P53野生型细胞U2OS, Catalase抑制了Azurin诱导的胞内ROS水平上调以及细胞凋亡,却对Azurin所致的GSH水平降低没有影响,这表明Azurin在U20S细胞中首先引发了GSH水平的下降,进而导致了氧自由基过氧化氢(H2O2)的积聚。GSH下降是先于ROS积聚发生的上游事件。
我们还采用超氧阴离子清除剂MnTBAP预处理P53野生型细胞U20S，发现其对Azurin诱导的胞内氧化应激、caspase活性均没有影响,这表明虽然超氧阴离子水平呈现上调,但其并未参与Azurin诱导的细胞凋亡。
同U20S细胞相比,P53突变型细胞MG63、p53缺失型SAOS-2经Azurin处理后,过氧化氢(H2O2)、超氧阴离子(O2-)、细胞外液GSH、细胞内GSH含量、谷胱甘肽过氧化物酶(GPX)活性均未见明显改变。
BSO抑制细胞内GSH合成,P53突变型MG63、p53缺失型SAOS-2细胞经BSO作用后,胞内GSH显著下降,氧自由基水平急剧增高,线粒体△Ψm明显降低,线粒体内膜脂质发生过氧化,Caspase活化,诱导细胞发生凋亡。BSO模拟了Azurin在U20S中的细胞毒性效应。
上述结果表明GSH耗竭、ROS的积聚在Azurin诱导的细胞凋亡中发挥着重要作用,并且氧化应激有可能受p53途径调控。
最后,我们观察了P53信号通路、氧化应激、线粒体损伤、细胞凋亡间的相互关系。
我们将报告基因质粒pp53-TA-luc和内参Renilla-luc瞬时共转染肺癌细胞株H1299(null). H322(mut)、A549(wt);肝癌细胞株Hep3B (null). Huh-7(mut)、 HepG2(wt);骨肉瘤细胞株SAOS-2(null)、MG-63(mut)、U20S (wt),荧光检测仪检测荧光素酶活性,我们发现Azurin增强了p53基因的转录活性。
试验进一步采用p53抑制剂PFT-α(pifithrinalpha,PFT-α)干扰试验,阻断U2OS的P53信号途径,结果显示PFT-α的预处理抑制了Azurin触发的GPX活性降低、GSH下降、ROS上调；线粒体膜电位△Ψm降低、内膜心磷脂氧化；Bax移位线粒体；细胞色素C、AIF释放进入胞质。这表明Azurin介导的氧化还原状态失衡、线粒体损伤受p53信号途径调控。
此外,实验结果还显示：在U2OS细胞中,NAC预处理抑制了Azurin上调的P53转录活性,而GSH耗竭剂BSO则在U2OS细胞中模拟了Azurin上调的P53转录活性,从而提示氧化还原状态的失衡正反馈激活了P53信号途径。
上述研究结果表明,P53信号通路及其调控的GSH代谢、ROS水平、线粒体途径,在Azurin诱导的靶细胞凋亡过程中扮演着重要角色。
我们选取骨肉瘤化疗常用药物如顺铂(Cisplatin, CDDP).氨甲喋呤(MTX)、阿霉素(ADM)和异环磷酰胺(IFO),在U20S、MG-63、SAOS-2细胞中,进行Azurin的药物增敏活性筛选,以验证小剂量Azurin联合低浓度化疗药物是否对骨肉瘤细胞的杀伤具有增敏效应?并就P53、ROS、GSH在其中的分子机制进一步探讨。
在顺铂、氨甲喋呤的药敏试验中,在U2OS细胞中,MTT显示,低剂量Azurin显著增加顺铂、氨甲喋呤的细胞毒性,联合给药具有协同效应。同时,也引发了GSH下降、ROS增加。还原剂NAC p53抑制剂PFT-α(pifithrinalpha,PFT-a)预处理U20S后,明显抑制了联合给药组所致的GSH降低、ROS上调以及细胞存活率的下降。而在P53功能缺失的其余二株MG-63、SAOS-2细胞中,联合给药组缺乏协同效应,同时,MG-63、SAOS-2胞内GSH、ROS水平也几乎没有改变。
以上数据表明了在Azurin联合顺铂、氨甲喋呤组的增敏效应中,P53及其调控的氧化还原状态失衡尤为关键。
在阿霉素、异环磷酰胺的药敏试验中,有趣的是,在三种MG-63、SAOS-2、 U2OS细胞中,小剂量Azurin都显著增强了阿霉素、异环磷酰胺的细胞毒性,在联合给药中,我们没有观察到GSH下降、ROS增加,还原剂NAC、p53抑制剂PFT-α(pifithrinalpha,PFT-α)的预处理对联合给药组所致的细胞存活率的下降也近乎没有影响,从而提示在MG-63、SAOS-2、U2OS中,Azurin对阿霉素、异环磷酰胺细胞毒性的增敏效应与P53、ROS途径无关。这表明在P53功能性缺失的肿瘤细胞中,Azurin也可对化疗药物的细胞毒性具有协同增敏效应,从而避免其局限于P53野生型肿瘤细胞。
此外,试验还发现对于人外周血单核细胞(PBMC)细胞,Azurin联合顺铂、氨甲喋呤、阿霉素、异环磷酰胺给药组的细胞毒性相对较小。这意味着,联合给药组既避免了单独给药时肿瘤细胞的获得性耐药,又使得其对正常体细胞的毒副反应大大降低。
结论
综上所述,在杨迪生教授、苗旭东等人的工作基础上,我们进一步探讨了P53、 ROS、线粒体途径在Azurin细胞毒性中的分子机制：Azurin增强p53转录活性↑→p53胞内水平↑→谷胱甘肽过氧化物酶(GPX)↓→GSH↓→ROS↑→线粒体内膜心磷脂氧化→线粒体膜电位(△Ψm)↓→Bax移位线粒体→细胞色素C、AIF释放进入胞质→激活线粒体途径→caspase活化→骨肉瘤细胞凋亡。P53基因调控的氧化还原状态失衡(GSH耗竭和ROS增高)在其中尤为关键。此外,通过P53依赖性或者非P53依赖性途径,Azurin与低剂量化疗药物如顺铂(Cisplatin, CDDP)、氨甲喋呤(MTX)、阿霉素(ADM)和异环磷酰胺(IFO)的联合给药具有协同效应,Azurin对上述化疗药物体外骨肉瘤细胞的细胞毒性具有增敏效应,其P53依赖性增敏机制与P53调控的氧化应激密切相关,而其不依赖于P53的增敏机制则与氧自由基水平无关。
本论文的研究结果将不仅有助于发现Azurin的可能作用靶点以及信号通路,还为Azurin的临床前期开发应用提供充分的实验依据,也为今后Azurin应用于临床骨肉瘤化疗的联合用药提供了安全有效的分子策略。
Backgroud:
Osteosarcoma because of its high degree of malignancy, early metastasis, clinical treatment with surgery combined with chemotherapy and radiotherapy treatments, but limited to the surgical treatment of high morbidity and poor function of the prosthesis, put the side effects of chemotherapy, advanced tumors easy to form a tolerance to chemotherapeutic drugs, vulnerable to relapse, re-treatment is also extremely difficult, and therefore lower long-term survival of osteosarcoma difficult to satisfactory efficacy, the limitations of traditional therapy makes osteosarcoma new therapies such as biological therapy, immunotherapy therapy, gene therapy, and microbial treatment of the purified product are increasingly becoming the focus of research and clinical and hotspots.
Yamada reported in recent years by Pseudomonas bacteria secrete copper-containing bacterial redox protein Azurin, can combine to form complexes with the P53protein, inhibition of P53degradation, thereby stabilizing the P53protein.It can be induced in vitro cell assays and in vivo tumor-bearing nude mice P53wild type mononuclear macrophages, melanoma cells, breast cancer cells, while P53mutation or deletion of melanoma cells in the rendered breast cancer cells, the cytotoxicity is greatly reduced, its cytotoxic obvious p53selectivity.Particularly critical in malignant melanoma cells, breast cancer cells in nude mice transplanted tumor, tumor size, and the wild-type Azurin can promote the degeneration of the transplanted tumors, inhibiting the growth of transplanted tumors tumor block induction of tumor cell apoptosis, while nude mice The body was found no significant side effects.This finding viable cells product purified protein the Azurin used in clinical tumor therapy to bring hope and prospects.
Zhejiang Second Orthopaedic Institute Professor Yang Disheng and Miao Xudong et al Azurin protein in human osteosarcoma cells to carry out a series of in vitro experimental study.The results are as follows:Azurin inhibited the growth of osteosarcoma induced P53wild type U2OS cell apoptosis, while normal liver L-02the P53mutant human osteosarcoma MG-63cells less toxic.Further study its molecular mechanisms:P53protein is play the key factors of the apoptotic effects the Bax mitochondrial shift of Bcl-2/Bax ratio downward, caspase-3,8increased activity, mitochondrial, extracellular Fas by pathway Azurin-induced the U2OS cell apoptosis molecular mechanisms are involved.
Tumor cells escape apoptosis caused apoptosis resistance (resistance to apoptosis) is one of the main reasons of tumor drug resistance, restore the system state level of intracellular reactive oxygen species (ROS) and glutathione (GSH) change is closely related to the apoptosis.
The wild-type p53gene is a tumor suppressor gene, the main function of p53is to arrest the cell cycle or induce apoptosis.There are three key steps in the p53-induced apoptosis signaling pathways:(i) oxidation reduction related gene transcription induced;(ii) the formation of reactive oxygen species (ROS) is formed; component (iii) mitochondrial oxidative degradation, and ultimately activation caspase leading to apoptosis.
In osteosarcoma chemotherapy drug toxicity reaction, while tumor recurrence rate was high due to repeated administration can easily acquire resistance, and therefore the long-term survival rate is lower.Studies have shown that the the ROS level differences related to the resistance of tumor cells to their cells.Through the regulation of intracellular GSH levels or elevated ROS levels can enhance the apoptosis-inducing effects of chemotherapy drugs.
Accordingly, we speculate Azurin raised p53transcriptional activity, triggered intracellular oxidation reduction state of imbalance through the mitochondrial pathway induction of apoptosis, and at the same time, Azurin of osteosarcoma tumor chemotherapy drug cis-platinum (Cisplatin, of CDDP), methotrexate chatter methotrexate (MTX). adriamycin (ADM) and ifosfamide (IFO) with chemotherapy sensitizing effect?And to explore the the p53, GSH, ROS in Azurin chemosensitizing effect of the specific mechanism of action.
Methods:
In our study, three different genotypes of P53osteosarcoma cell line (p53wild-type U2OS, p53mutant MG-63, p53-deficient SAOS-2) and a p53wild-type normal Human peripheral blood mononuclear cells (PMBCs), by MTT colorimetric assay of cell viability, application of Annexin V and PI double staining flow cytometry apoptosis rate Annexin V positive cell (%) by flow cytometry, fluorescence microscopy detection of mitochondrial membrane potential (ΔΨm) and mitochondrial inner membrane heart phospholipids oxidation by immunofluorescence techniques, laser confocal subcellular protein separation Cytochrome C (cytochrome C) Western blot analysis of mitochondrial outer membrane protein, AIF (apoptosis-inducing factor) release into the cytoplasm, cytoplasmic Bax shift mitochondria, Western blot analysis of mitochondrial core apoptotic protein Bcl-xL of MCL-1, XIAP, survivin, Bak, Bid, tBid of expression by flow cytometry reactive oxygen species (H202), superoxide anion (O2-) and nitric oxide (NO), through the the microplate colorimetric detection intracellular GSH, extracellular fluid GSH, glutathione peroxidase (GPX), GSSG (oxidized form of GSH), glutathione reductase (GR) level, pp53-TA-luc reporter plasmid and the internal reference Renilla-luc transiently co-transfected osteosarcoma cells SAOS-2(null), MG-63(mut), U2OS (wt), the fluorescence detector detects the above cell luciferase activity, namely the transcriptional activity of the p53gene.And mitochondrial membrane PT pore inhibitor cyclosporine A (CsA); reductant Catalase, NAC, MnTBAP; GSH depleting agent BSO; P53signal blocking agent PFT-a verified from two positive and negative direction to clear mitochondria ROS, P53specific mechanism of action.
Result:
First, we observed Azurin P53pathway induced tumor cell apoptosis.
Further clear of Azurin the P53selectively kill tumor mechanism, we have selected different P53genotype background of tumor cells in other tissue sources:53people carrying wild-type P hepatoma cell line HepG2cell lines, lung cancer cell line A549, flesh and blood tumor cell line U2OS; P53mutant lung cancer cell line H322, hepatoma cell line Huh-7cells, osteosarcoma cell line MG-63; render P53deletion lung cancer cell lines HI299hepatoma cell line Hep3B cells, osteosarcoma SAOS-2cell lines; carrying the wild-type P53normal human peripheral blood the monocytes (PMBCs).Azurin proliferation of these cell lines by MTT assay.
By MTT assay, we found that the tumor cells in the background of P53gene in lung cancer, liver cancer, osteosarcoma cell lines, Azurin also significantly inhibited with wild-type P53the HepG2, A549, U2OS cells proliferation activity, whom In contrast, in the cells of P53functional inactivation AZURIN cell toxicity is significantly weakened.In these cells, Azurin perform equally obvious P53selective cytotoxic effect, simultaneously less toxic of normal PMBCs cells.
Azurin treatment in p53wild-type U2OS cells, caspase-3activity was significantly increased, start caspase cascade, PARP occurred shear induced apoptosis.Conversely, p53mutated MG-63, p53-deficient SAOS-2cell lines Azurin upregulation of caspase-3kinase activity, Annexin V positive cell (%) were significantly weakened; trials further a P53signal blocker PFT-a preincubation U2OS cells, were observed on the the Azurin role Cleaved-PARP protein kinase caspase-3activity, Annexin V positive cell (%), MTT indicators, we find that PFT-a almost completely inhibited Azurin induced Caspase-3activation, PARP a shear, Annexin V positive (%) of the raised; thus confirming the molecular mechanism of Azurin apoptosis presented P53-dependent manner.
Secondly, we detect the the Azurin induced mitochondrial damage.
P53wild-type cells U2OS after Azurin role, JC-1red and green fluorescence ratio decreased mitochondrial membrane potential (ΔΨm) happened; mitochondrial NAO fluorescence intensity was significantly reduced, and reduce mitochondrial endometrial cardiolipin content endometrial produce a shift gathered mitochondrial outer membrane lipid peroxidation, causing increased permeability of the outer mitochondrial membrane; cytoplasmic protein Bax monomer targeted protein conformational change occurred oligomerization to form across the mitochondrial outer membrane pore, lead to mitochondrial further increase in membrane permeability.Bcl-2family inhibiting apoptotic protein Bcl-xL and MCL-1, XIAP protein expression levels of self-8h time point reduced to promote the expression of apoptotic protein Bak presents a time-dependent increase, while the truncated Bid (tBid) from the4h time to point began to appear, the24h time point tBid the protein expression levels peak of Bid Protein shear.Cytochrome C combined with cardiolipin be uncoupling between the inner and outer mitochondrial membrane protein Cytochrome C (Cytochrome C), AIF (apoptosis inducing factor) protein pore massive release into the cytoplasm, start caspase cascade activation of downstream effector caspase-3, AIF thus translocation nucleus and DNA binding in the nuclear cause staining quality gathering and fracture.
It is noteworthy that in p53mutant MG-63, p53-deficient SAOS-2cells and normal human peripheral blood mononuclear cells (PMBCs) Azurin treatment, mitochondrial membrane potential ΔΨm, mitochondrial inner membrane center phospholipid content did not change significantly, suggesting that p53signaling pathways Azurin-induced mitochondrial damage.
To clear the mitochondrial pathway Azurin induced apoptosis, mitochondrial membrane PT pore inhibitor cyclosporine A (CsA) pre-incubated U2OS cells, CsA significantly inhibited the the Azurin enhanced caspase-3activity, Cleaved-PARP The upregulation of the protein, and apoptosis.But raised on Azurin induced intracellular ROS levels, GSH levels decreased almost no effect, suggesting that mitochondrial channel opening events later, in the redox system imbalance occurs downstream events.
Again, we observed Azurin-induced cell redox state of imbalance.
Azurin in a P53wild-type U2OS cells, quickly lead to the DCF (H2O2), ethidium (O2-) significant upregulation of the fluorescence intensity, but then the fluorescence intensity of the DAF-FM (NO) does not affect, i.e. The Azurin mainly caused too hydrogen peroxide (H2O2) and superoxide anion (O2-) of these two kinds of radicals rise, but then the nitric oxide (NO) no effect.
Meanwhile, we observed the Azurin U20S cells GSH reduction system, we found Azurin suppression Synthesis and enzyme activity of glutathione peroxidase (GPX), oxidized GSSG content increased greatly reduces the antioxidant capacity of the GSH reduction system, to induce GSH release into the extracellular fluid, further depletion of intracellular GSH, promote hair redox system imbalance.
Application of GSH precursor antioxidants NAC pretreatment p53wild-type U20S cells, we found that NAC significantly inhibited the Azurin induced intracellular GSH level reduced and ROS upward, also prevent a decline in mitochondrial membrane potential and intimal Azurin priming heart phospholipid peroxidation, and eventually suppressed caspase activation mediated apoptosis.Which prompted Azurin triggered intracellular redox state imbalance between mitochondrial damage occurs before the upstream events.
Test further reducing agent, hydrogen peroxide (H2O2) the enzyme Catalase pretreatment P53wild-type cells U20S Catalase suppression Azurin induced intracellular ROS levels increase, and apoptosis, but then the Azurin due to the reduced GSH levels did not impact, which indicates that the decline in the level of GSH Azurin first initiator in U2OS cells, leading to oxygen radicals of hydrogen peroxide (H2O2) accumulation.GSH decreased upstream events prior to ROS accumulation occurred.
We also used the superoxide anion scavenger MnTBAP pretreatment P53wild type cells U2OS the the Azurin induced intracellular oxidative stress, caspase activity had no effect, suggesting that superoxide anion levels presented raised, but did not participate in The Azurin induced apoptosis.
Compared with U2OS cells, P53mutant cells of MG63of p53-deficient SAOS-2after Azurin processing, hydrogen peroxide (H2O2), superoxide anion (O2-) cell extracellular fluid GSH intracellular GSH content, glutathione Gan peptide peroxidase (GPX) activity showed no significant change.
The BSO inhibition of intracellular GSH synthesis P53mutant of MG63and p53-deficient SAOS-2cells after BSO role of intracellular GSH was significantly decreased, and a sharp increase in the level of oxygen free radicals, significantly decreased the ΔΨm, mitochondrial inner membrane lipid peroxidation occurred, Caspase activation and induce apoptosis.The BSO analog Azurin in U2OS cytotoxic effect.
The above results indicate that GSH depletion, accumulation of ROS-induced apoptosis in Azurin plays an important role, and oxidative stress may be affected by the p53pathway regulation.
Finally, we observed p53signaling pathway, the relationship between oxidative stress, mitochondrial damage, apoptosis.
We will pp53-TA-luc reporter plasmid and the internal reference Renilla-luc instantaneous co-transfected lung cancer cell lines H1299(null), H322(mut), A549(wt); hepatoma cell line Hep3B (null), Huh-7(mut was), HepG2(wt); osteosarcoma cell line, SAOS-2(null), MG-63(MUT), U2OS,(by weight), the fluorescent detector detecting the luciferase activity, we found to The Azurin enhance the transcriptional activity of the p53gene.
Further test p53inhibitor PFT-α (pifithrinalpha, PFT-α) interference test, blocked U2OS P53signaling pathways, and results showed that PFT-a pretreatment inhibits Azurin triggered GPX activity decreased, GSH decreased, ROS raised; mitochondrial membrane potential ΔΨm lower endometrial cardiolipin oxidation; the Bax shift mitochondria; cytochrome C, AIF release into the cytoplasm.This indicates that the redox state of imbalance Azurin mediated mitochondrial damage by the p53signaling pathway regulation.
In addition, the results also show that:U2OS cells, NAC pretreatment inhibited Azurin upregulation of p53transcriptional activity, GSH depletion agent BSO in U2OS cells to simulate the the Azurin raised p53transcriptional activity, suggesting the redox state of imbalance Positive feedback activation of the p53signaling pathway.
The results of the study show that the P53signaling pathway and its regulation of GSH metabolism, ROS levels, mitochondrial pathway in the Azurin induced target cell apoptosis process plays an important role.
We selected the commonly used drugs such as osteosarcoma chemotherapy Cisplatin (Cisplatin, CDDP), methotrexate (MTX), adriamycin (ADM) and Ifosfamide (IFO), U2OS MG-63, SAOS-2cells, Azurin drug-sensitizing activity screening to verify that small doses Azurin combined with low concentrations of chemotherapy drugs osteosarcoma cells destruction sensitizing effect?And P53, ROS, GSH in which to further explore the molecular mechanisms.
Susceptibility testing of cisplatin, methotrexate, U2OS cells, MTT showed that low-dose the Azurin significant increase cisplatin, methotrexate cytotoxicity, administered in combination have a synergistic effect.At the same time, it also raised GSH decline, ROS increase.Reducing agent NAC, p53inhibitor PFT-a (pifithrinalpha, PFT-a) after pretreatment U2OS significantly inhibited due to the co-administered group of GSH decreased ROS raised as well as the reduction in cellular viability.In P53loss of function of the remaining two MG-63, SAOS-2cells, combination group lack of synergy effects, the MG-63, SAOS-2intracellular GSH of ROS levels are almost no change.
These data suggest that the P53and its regulation of the redox state of imbalance is particularly critical in Azurin combination with cisplatin, methotrexate group sensitizing effect.
Doxorubicin, ifosfamide susceptibility testing, it is interesting that three kinds of MG-63, SAOS-2, U2OS cells, small doses of Azurin significantly enhanced doxorubicin, ifosfamide cytotoxicity, co-administration, we have not observed GSH decline, ROS increases, reducing agent NAC p53inhibitor PFT-a (pifithrinalpha, PFT-a) pretreatment cell viability due to the co-administered group decline in almost no effect, which prompted the MG-63, SAOS-2, U2OS, Azurin doxorubicin, different sensitizing effect of cyclophosphamide cytotoxicity P53, ROS pathway unrelated.This shows that in the p53functional deletion of tumor cells, Azurin also be synergistic sensitizing effect of cytotoxic chemotherapy drugs, thus avoiding its limited to P53wild type tumor cells.
In addition, the tests also found that the cells of the human peripheral blood mononuclear cells (PBMC), AZURIN cisplatin, methotrexate, adriamycin, ifosfamide administration group cytotoxic relatively small.This means that the co-administration group only avoids acquired drug resistance of tumor cells when administered alone, but also makes its toxic side effects on normal cells greatly reduced.
Conclusion:
In In summary, on the basis of the work of Professor Yang Disheng, Miao Xudong et al, we further investigate the P53, ROS, mitochondrial pathway of the molecular mechanisms of cytotoxicity in Azurin:Azurin enhanced p53transcriptional activity↑→p53intracellular levels→GSH↓of ROS, the↑→glutathione peroxidase (GPX)↓↑→mitochondrial oxidation of endometrial cardiolipin→mitochondrial membrane potential (ΔΨ m)↓→Bax shift mitochondrial→cytochrome C, AIF release into cell the quality→activation of the mitochondrial pathway→caspase activation→apoptosis of osteosarcoma.Redox state of the P53gene regulatory imbalance (GSH depletion and increased ROS) in which is particularly critical.In addition, the P53-dependent or non-dependent pathway of P53Azurin with low doses of chemotherapy drugs such as cisplatin (Cisplatin, CDDP), methotrexate methotrexate (MTX), adriamycin (ADM) and ifosfamide (IFO) to the United The drugs have a synergistic effect, Azurin sensitizing effect of the chemotherapy drugs osteosarcoma cells in vitro cytotoxic, oxidative stress is closely related to its P53-dependent the sensitizing mechanism with P53control, which does not depend on the P53sensitizing mechanism has nothing to do with the level of oxygen free radicals.
The findings of this paper will not only help to identify the Azurin the possible targets of signaling pathways also provide sufficient experimental evidence of Azurin the preclinical development and application, and also for the future Azurin used in clinical osteosarcoma chemotherapy combination provides a safe and effective molecular strategies.
引文
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