以蛋白酶体为靶点的地钱素M诱导前列腺癌细胞死亡的机制研究
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摘要
前列腺癌(prostate cancer, PCa)是欧美国家老年男性最常见的恶性肿瘤,其死亡率仅次于肺癌。虽然我国前列腺癌的发病率明显低于西方国家,但随着人口老龄化、饮食结构的改变等,我国前列腺癌发病率,特别是晚期前列腺癌的发病率呈显著上升趋势。据预计10年后,前列腺癌在我国的发病率可能会超过60-70/10万,已成为威胁我国老年男性健康的恶性疾病之一。
前列腺癌是激素依赖性肿瘤,因此内分泌治疗是治疗前列腺癌的首选标准方案。然而绝大多数前列腺癌患者经内分泌治疗1-2年后易发展为激素难治性前列腺癌(Hormone Refractory Prostate Cancer,HRPC),对雄激素撤除不敏感,抵抗凋亡并伴随侵袭转移,预后差,死亡率高,目前临床尚无有效的治疗方法。由于HRPC的演变是复杂的多因素、多信号途径参与的过程,其机制尚不完全清楚,因此寻找有效的治疗靶点及机制,产生多种途径的协同效应,是治疗前列腺癌的有效策略之一。
第一部分地钱素M抑制蛋白酶体的活性,诱导细胞内质网应激和凋亡
泛素-蛋白酶体系统(Ubiquitin-proteasome system, UPS)是真核生物细胞内重要的蛋白质质量控制体系之一。UPS通过将小分子蛋白泛素与靶蛋白的赖氨酸残基共价结合,从而将其泛素化。泛素标记的蛋白被蛋白酶体特异性地识别并迅速降解。UPS通常降解受损变性蛋白、短寿命的调控蛋白等,对于细胞周期运转、凋亡与分化、信号转导等生理过程起着重要的调控作用。恶性肿瘤细胞内蛋白代谢紊乱,蛋白酶体活性升高,UPS调节失衡,使得肿瘤细胞更加依赖UPS,以维持细胞内稳态。因此,肿瘤细胞对蛋白酶体抑制剂更加敏感,蛋白酶体也成为肿瘤治疗的新靶点,开发针对UPS的抑制剂在肿瘤治疗中具有广阔的前景,而天然化合物以其独特的结构、多样的生物学活性更是备受关注。
双联苄(Bisbibenzyls)是苔藓植物中分离得到的一类大环多酚类化合物,因联苄之间的连接方式不同、取代基不同而具有结构新颖、多样的特点,具有抗真菌、抗炎、抗肿瘤等多种生物活性。本课题组前期研究发现双联苄类化合物地钱素M(Marchantin M,Mar)显著抑制前列腺癌细胞的增殖、促进其凋亡。由于地钱素M的结构与茶多酚的功能区域相似,而茶多酚已被证明是蛋白酶体的不可逆抑制剂。我们推测地钱素M可能具有抑制蛋白酶体活性的功能。为此,我们研究了地钱素M对重组纯化的20S蛋白酶体、前列腺癌细胞内蛋白酶体活性的影响,并对其生物学效应进行了分析,取得了以下研究结果：
一、地钱素M显著抑制蛋白酶体活性,是新的可逆性蛋白酶体抑制剂
1.地钱素M显著抑制蛋白酶体胰凝乳样蛋白酶(ChT-L)和肽基谷氨酰多肽解酶(PGPH)活性。Mar显著抑制重组纯化的20S蛋白酶体中ChT-L和PGPH活性,呈剂量依赖性。其半数抑制率分别为6.99和5.33μM。但不影响胰蛋白酶样活性。细胞实验表明,地钱素M对激素非依赖性前列腺癌PC3,DU145细胞和激素依赖性前列腺癌LNCaP细胞内26S蛋白酶体显示同样的抑制作用,且呈时间和剂量依赖性。表达差异谱基因芯片数据显示,地钱素M并不影响UPS途径中各组分的基因表达。
2.地钱素M是新的可逆性蛋白酶体抑制剂。通过Autodock vina软件分析发现,地钱素M能与蛋白酶体的β1(PGPH)和β5(ChT-L)亚基结合。二者主要依靠氢键、疏水键等非共价键相互作用,其自由能分别为：-7.4kcal/mol和-6.5kcal/mol。林-贝氏双倒数作图法表明,地钱素M对蛋白酶体的抑制呈现较典型的非竞争性抑制的方式。上述结果表明,地钱素M是一种新的蛋白酶体可逆的抑制剂。
3.地钱素M抑制蛋白酶体活性,导致前列腺癌细胞内多泛素化蛋白累积,且呈时间、剂量依赖性。
二、地钱素M抑制蛋白酶体活性,导致内质网相关蛋白降解途径(ERAD)受阻,诱导内质网应激和细胞凋亡
1.地钱素M介导的蛋白酶体抑制,抑制ERAD,诱导内质网应激。地钱素M处理前列腺癌细胞后,导致经ERAD降解的蛋白如突变蛋白CFTRAF508、SPCA4在内质网堆积；电镜下观察到内质网肿胀,激光共聚焦结果显示高浓度的地钱素M可引起内质网空泡化；Western blotting结果表明,内质网伴侣蛋白GRP78表达显著上调、PERK及其下游eIF2α磷酸化水平升高；定量PCR分析显示应激分子ATF6.ATF4及其靶基因ATF3表达显著增加；IRE1激活并剪接XBP1的mRNA,激活XBP1。上述结果表明,地钱素M抑制蛋白酶体活性导致内质网内未折叠或错误折叠的蛋白累积,诱导内质网应激。
2.地钱素M诱导前列腺癌细胞凋亡。地钱素M呈剂量依赖性抑制激素依赖、激素非依赖性前列腺癌细胞凋亡,且使激素依赖性细胞更易凋亡。Western blotting结果表明,地钱素M可激活内质网特异caspase4,从而激活caspase3,诱导凋亡。
3.Caspase非依赖途径参与地钱素M诱导的细胞凋亡。在激素依赖的前列腺癌LNCaP细胞中,Caspase抑制剂z-VAD-fmk逆转绝大多数地钱素M诱导的细胞死亡,而在激素非依赖的前列腺癌PC3和DU145细胞中,z-VAD-fmk只能部分部分逆转地钱素M介导的细胞死亡。上述结果表明,地钱素M主要通过caspase依赖的途径诱导激素依赖性前列腺癌细胞凋亡,而通过caspase依赖和非依赖机制诱导激素非依赖性前列腺癌细胞死亡。
第二部分地钱素M上调应激基因ATF3的表达而抑制激素依赖性前列腺癌细胞中AR的功能
雄激素受体(Androgen receptor,AR)为核受体型转录因子。当雄激素(如睾酮、二氢睾酮)与AR结合后,引起AR构象改变,使AR与热休克蛋白解离、活化而转移至核内,并形成同源二聚体与DNA分子中雄激素受体应答元件(AREs)结合,诱导下游靶基因如前列腺特异性抗原(prostate specific antigen,PSA)等基因的转录表达,促进细胞生长和存活。因此,AR信号通路不仅在维持前列腺的正常发育、功能发挥中起主要作用,也是激素依赖性前列腺癌增殖的关键途径,而且在激素非依赖性转化过程中起决定性作用,并在约1/3的HRPC中持续性高表达。AR基因扩增、突变、过表达以及AR激素非依赖性激活,均可引起AR功能的异常活化,是前列腺癌内分泌治疗失败、侵袭转移、抵抗凋亡等恶性表型的重要机制之一。因此,有效抑制前列腺癌中AR的表达或和功能一直是倍受关注的研究课题。
ATF3(activating transcription factor3)作为转录因子ATF/CREB家族成员,通过其蛋白碱性区的亮氨酸拉链结合到特异DNA的ATF/CREB顺式作用元件上,调控基因表达。当DNA损伤、毒素暴露、氧化应激以及内质网应激时,ATF3可被迅速诱导表达,以维持细胞在应激条件下的基因组完整性和内稳态。然而,在持续或过强的应激状态下,ATF3可介导细胞凋亡。由于ATF3蛋白中的亮氨酸拉链可介导ATF3与多种蛋白相互作用。本课题第一部分研究结果显示,地钱素M诱导内质网应激,显著增加ATF3表达,且主要以caspase依赖的凋亡形式诱导激素依赖性前列腺癌细胞LNCaP死亡。由于AR是LNCaP细胞增殖存活的关键调控因子,我们研究了ATF3高表达对AR表达和功能的影响以及与LNCaP凋亡的关系,结果如下：
一、地钱素M通过内质网应激信号PERK/eIF2α/ATF4上调ATF3的表达
1.地钱素M在转录水平上调ATF3的表达。地钱素M显著增加前列腺癌细胞中ATF3启动子活性,促进其转录。利用MatInspector软件分析ATF3启动子区转录因子的结合位点,发现-22/-18区存在ATF的结合位点；通过构建ATF3启动子及其系列截短体和ATF结合区的点突变和缺失突变的报告基因载体。双荧光素酶检测结果表明,ATF结合位点对地钱素M诱导的ATF3转录激活至关重要。
2.地钱素M诱导的ATF3表达上调主要受内质网应激信号PERK/eIF2α/ATF4的调控。通过显性负相关表达载体抑制PERK的活性或siRNA下调ATF4的表达均能显著下调ATF3的表达,同时促进细胞存活。
二、地钱素M诱导的ATF3高表达促进LNCaP细胞凋亡
1.抑制ATF3的表达促进细胞存活。利用显性负相关或siRNA技术抑制ATF3的表达,细胞增殖实验结果显示LNCaP细胞的存活率显著提高；AnnexinV-FITC/PI双染结果表明,下调ATF3后,凋亡细胞显著减少。
2.ATF3通过caspase途径诱导细胞凋亡。Western-blotting结果显示,降低ATF3的表达,可抑制caspase3的活化,使caspase3的底物PARP剪切减少。上述结果表明,地钱素M上调ATF3的表达促进LNCaP经caspase依赖的途径凋亡。
三、高表达的ATF3通过蛋白间相互作用抑制AR的功能、促进LNCaP细胞凋亡
1.地钱素M显著上调ATF3蛋白水平。Western-blotting结果显示,地钱素M显著提高ATF3的蛋白表达,而且在激素依赖性前列腺癌细胞LNCaP中的诱导作用远远高于激素非依赖性前列腺癌细胞PC3和DU145。
2.ATF3与AR相互作用抑制其功能。免疫共沉淀结果显示,地钱素M诱导的ATF3与AR结合。在LNCaP细胞中过表达ATF3显著下调AR的靶基因PSA的启动子活性及其mRNA水平,其它AR调控的靶基因,如TMPRSS23和NKX3.1等表达均明显下调。利用siRNA干扰ATF3后,上述AR靶基因都有不同程度的恢复表达。当AR缺失的PC3细胞共转染AR,PSA启动子和不同剂量的ATF3表达载体后,PSA启动子活性随ATF3表达量的增加而降低。以上结果说明地钱素M诱导的ATF3通过与AR结合而抑制其功能,从而诱导LNCaP细胞凋亡。
第三部分地钱素M诱导激素非依赖性前列腺癌细胞自噬性死亡
在激素非依赖性前列腺癌细胞PC3和DU145中,阻断caspase依赖的细胞凋亡,只能部分逆转地钱素M诱导的细胞死亡,提示caspase非依赖的细胞死亡机制参与地钱素M诱导的细胞死亡过程。
已有研究表明,抑制蛋白酶体活性不仅能够促进肿瘤细胞凋亡,还可通过多种机制诱导细胞自噬(autophagy)。自噬是真核细胞内除UPS外,另外一种蛋白质质量控制系统,主要通过自噬泡包裹细胞胞液的内容物,包括衰老或受损的亚细胞器如线粒体、长寿命蛋白、入侵的微生物等运送至溶酶体降解,降解产物可以被细胞再次利用。当细胞面临各种刺激、应激时,自噬可被诱导增强,可通过出现的自噬泡、自噬相关基因的表达等方法进行检测。自噬对肿瘤细胞具有双重作用,在肿瘤发生的不同阶段,自噬所起的作用不同。在正常条件下,基础性自噬可清除细胞内可能产生毒性作用的受损成分,抑制细胞的恶性转化,预防肿瘤发生。当肿瘤细胞面临乏氧、营养缺乏以及化疗药物时,自噬的激活会提供能量、营养物质等而对肿瘤细胞起到保护作用,有利于细胞存活。然而多种应激而产生持续、过度的自噬时,也会导致细胞死亡,称为自噬性细胞死亡(autophagic cell death),即Ⅱ型程序性细胞死亡。地钱素M处理的前列腺癌细胞,不仅使自噬的标志性蛋白LC3的表达增加,而且促进LC3I向LC3Ⅱ转变,表明地钱素M能诱导前列腺癌细胞产生自噬。因此,我们研究了地钱素M诱导的自噬在前列腺癌细胞死亡过程中的作用,并对其信号通路进行了探讨,取得了以下研究成果：
一、地钱素M抑制蛋白酶体活性,几乎同时诱导自噬的发生
1.地钱素M抑制蛋白酶体活性伴随着内质网应激和细胞自噬。时相分析结果显示,地钱素M处理细胞1h时,蛋白酶体活性显著下降,而在处理2h后内质网伴侣蛋白GRP78的表达显著上调、自噬标志性蛋白LC3Ⅱ的表达开始上调并持续增强至48h。此结果表明,地钱素M抑制蛋白酶体活性后,随即引起内质网应激和细胞自噬。
2.地钱素M显著诱导细胞产生自噬。LC3Ⅱ的表达升高提示该化合物可使细胞产生自噬。透射电镜的结果明确显示,地钱素M使激素非依赖性前列腺癌细胞中自噬泡和自噬溶酶体明显增多,为自噬提供直接证据。激素非依赖性前列腺癌细胞瞬时转染GFP-LC3表达载体或利用GFP-LC3稳转的U87细胞株,均可观察到地钱素M极显著地诱导LC3聚集、产生聚集荧光斑点。
3.地钱素M诱导自噬流量的增加。基因芯片和定量PCR结果表明,地钱素M在转录水平诱导LC3基因表达升高,而其它自噬相关基因如Atg5、Atg7、Beclin1等表达几乎没有变化。Western blotting结果显示地钱素M不仅上调LC3的蛋白水平,而且促进LC3I向LC3Ⅱ转变,并引起自噬底物p62下降。利用氯喹阻断自噬泡和溶酶体融合进一步增加地钱素M诱导的LC3Ⅱ的累积,而利用自噬抑制剂3-MA则显著抑制地钱素M诱导的LC3荧光斑点的聚集。这些结果表明地钱素M抑制蛋白酶体活性后,迅速诱导内质网应激和细胞自噬,显著增加自噬流量。
二、地钱素M诱导激素非依赖性前列腺癌细胞自噬性死亡
1.自噬抑制剂逆转地钱素M诱导的细胞死亡。自噬抑制剂3-MA显著下调地钱素M诱导的LC3Ⅱ的表达,同时促进细胞存活,降低地钱素M介导的细胞死亡。利用E-64D/Pepstatin A抑制溶酶体酶的活性,使地钱素M诱导的LC3Ⅱ的表达略有增加,但依然显著增加细胞的存活,逆转地钱素M诱导的细胞死亡。
2.抑制自噬相关基因逆转地钱素M诱导的细胞死亡。利用siRNA特异性的降低Atg5、Atg7、LC3的表达,均可显著抑制LC3Ⅱ的表达或增加p62的表达而抑制自噬,同时显著逆转地钱素M诱导的细胞死亡,增加细胞存活率。然而干扰Beclinl后并不影响地钱素M介导的细胞死亡。上述结果表明,地钱素M诱导的自噬为自噬性死亡。
3.抑制自噬和caspase凋亡途径完全逆转地钱素M诱导的细胞死亡。由于caspase抑制剂只部分逆转地钱素M诱导的激素非依赖性前列腺癌细胞凋亡,采用siRNA下调Atg5的表达抑制自噬、同时联用caspase抑制剂,几乎完全逆转地钱素M诱导的激素非依赖性前列腺癌细胞死亡。因此,白噬性细胞死亡和caspase依赖的细胞凋亡并存于地钱素M的细胞毒作用。
三、地钱素M通过内质网应激信号PERK/eIF2α和PI3K/Akt/mTOR途径诱导自噬
1.地钱素M通过下调Akt1的表达抑制PI3K/Akt/mTOR途径诱导自噬。Westernblotting结果显示,地钱素M首先下调phospho-PDK1、phospho-Akt以及总Akt蛋白的表达水平,随后降低phospho-mTOR和phospho-p70S6K的水平,表明地钱素M通过抑制PI3K/Akt/mTOR途径而诱导自噬。定量PCR结果显示,与Akt2、Akt3相比,地钱素M极显著地抑制Akt1的mRNA水平。持续活化的Aktl表达载体能够显著逆转地钱素M诱导的LC3Ⅱ表达及细胞死亡,而野生型Aktl无此作用。此结果表明,地钱素M通过下调Akt的表达、抑制PI3K的活性,使Akt磷酸化水平下降,从而使mTOR失活而诱导自噬。
2.地钱素M诱导的内质网应激信号促进白噬。利用JNK抑制剂阻断JNK/c-Jun信号并不影响地钱素M诱导LC3Ⅱ表达及细胞死亡,而转染显性负相关的PERK表达载体或siRNA抑制PERK,阻断内质网应激信号PERK/eIF2α,地钱素M诱导的LC3Ⅱ表达显著下调、并部分逆转其诱导的细胞死亡。上述结果表明,内质网应激信号PERK/eIF2α参与了地钱素M诱导的自噬及细胞死亡过程。
第四部分结论及创新点
一、结论
1.双联苄化合物地钱素M抑制蛋白酶体活性,是一新型可逆性蛋白酶体抑制剂。
2.地钱素M通过抑制蛋白酶体活性、阻断内质网相关的蛋白降解途径而诱导内质网应激,促进前列腺癌细胞死亡。
3.地钱素M通过诱导内质网应激、促进应激转录因子ATF3的高表达；而高表达的ATF3通过与AR发生蛋白间相互作用抑制AR功能,从而诱导激素依赖性前列腺癌细胞主要发生细胞凋亡。
4.地钱素M通过诱导内质网应激和细胞自噬,促进激素非依赖性前列腺癌细胞发生caspase依赖的凋亡和自噬性细胞死亡
二、创新点与不足之处
1.首次报道双联苄化合物地钱素M靶向蛋白酶体、是一新型的可逆性蛋白酶体抑制剂。由于不可逆性蛋白酶体抑制剂易产生较大毒性,因此地钱素M的作用特点,使其成为很有研究价值的天然产物。
2.首次报道地钱素M可通过应激蛋白ATF3而抑制AR的功能,进而促进细胞凋亡。由于LNCaP细胞表达的是突变型AR,地钱素M的这种抑制作用将有益于AR持续活化的前列腺癌的治疗。但地钱素M对野生型AR的作用有待于进一步研究。
3.首次报道地钱素M可诱导激素非依赖性前列腺癌自噬性细胞死亡。由于大多数化疗药物如紫杉醇等可引起肿瘤细胞产生自噬性保护机制,削弱其抗肿瘤效果,而且激素非依赖性前列腺癌细胞比激素依赖性细胞抗凋亡能力强,对药物敏感性差。地钱素M对激素依赖、非依赖前列腺癌细胞的IC50值相近,且可引起自噬性细胞死亡,这些特点使地钱素M是很有潜力的抗肿瘤药物。但该化合物的动物药效学实验尚需进行。
Prostate cancer is the most common nonskin cancer in old men in the Western world. While improved early detection significantly decreased mortality, PCa still remains the second leading cause of cancer-related death in Western men. Although the incidence of prostate cancer was lower than the Western countries', in recent years confirmed PCa, especially for advanced or metastatic PCa, represents an obviously rising trend in China with the aging and the diet habit changing.
PCa is initially androgen-dependent with complicated pathogenesis. In addition to genetic factors, the incidence of prostate cancer is related with high levels of androgens, race, diet and lifestyle. PCa depends largely on androgen receptor (AR) signaling for growth and maintenance. Androgen deprivation therapy has become the standard first-line treatment for advanced hormone naive PCa. Unfortunately, the development of hormone-refractory prostate cancer (HRPC) has been observed to occur within a few years after hormonal deprivation therapy and is associated with poor prognosis, high apoptosis-resistance and high mortality. There are few efficacious treatment options available for curing, or even improving the survival and quality of life in patients with HRPC. Complicated factors and signals involves in the development of HRPC. Nevertheless, the underlie mechanism of PCa is still remains elusive. Therefore, novel strategies targeting the molecular basis of PCa progression and producing synergies of different signals are highly desirable.
Part I Marchantin M inhibits the activities of the proteasome and triggering ER stress and apoptosis
Ubiquitin-proteasome system (UPS) is one of protein quality control system in eukaryotic cells. UPS works mainly through ubiquitination of the target protein and degradation of abnormal proteins and short-lived protein by proteasome. UPS plays an essential role in cell proliferation, cell cycle regulation, immune response, signal transduction and cell death. Recent studies have validated that proteasome activities in malignant tumors are much higher than that in non-malignant cells, conferring tumor cells more dependent on the UPS to maintain cellular homeostasis. Therefore, tumor cells are more sensitive to UPS inhibitors. UPS has gradually become a new target for cancer therapy, and Reagents targeting at proteasome are expected to become promising anticancer drugs. Natural products have gained much attention in their unique structures and diverse bioactivities.
Macrocyclic bisbibenzyls are a series of phenolic natural products isolated from liverwort, which exert a variety of biological activities, including antifung, antioxidation and cytotoxicity. Our previous research demonstrates its inhibitory effect on PCa cells via activation of caspase pathway.
Based on the nature of its structure, Marchantin M (Mar) was somewhat similar to tea polyphenols, bearing with phenol groups which may act as tyrosine mimic binding to β5subunit of proteasome. We speculate that Marchantin M may be a novel proteasome inhibitor. Therefore, we discuss the inhibitory effect of Marchantin M on proteasome in prostate cancer cells.
一、Mar is a novel reversible inhibitors targeting proteasome activities
1. Mar dramatically inhibits ChT-L and PGPH activities of proteasome.
Mar inhibits ChT-L and PGPH activities of proteasome of20S proteasome in a dose-dependent manner in vitro. The IC50values for ChT-L and PGPH were6.99and5.33μM, respectively. However, Marchantin M hardly inhibited Try-L activity of the 20S proteasome. The Similar results were observed in Mar-treated PCa cells. Analysis of gene chip shows that Mar inhibits proteasome activities rather than suppresses expression of UPS components. The results indicate Mar is a novel inhibitor.
2. Mar is a reversible inhibitor of proteasome.
Since the ChT-L and PGPH activities were mediated by the β5and β1subunits of proteasome, we docked the Mar molecule into β5and β1subunits to investigate their interactions by autodock vina. The docking results revealed that Mar bound to P5and β1subunits at the active sites with a conformation suitable for proteasome inhibition. The docked free energy was-7.4kcal/mol and-6.5kcal/mol, respectively. The docking images also indicated that Marchantin M interacted with β1and β5subunits through hydrogen and hydrophobic interactions. Lineweaver-Burk double-reciprocal plots for the PGPH activity displayed characteristics of non-competitive inhibition, the Km value was determined to be48.52μM. The data clearly demonstrates that Mar is a novel reversible inhibitor of proteasome.
3. In accordance with proteasome inhibition, Marchantin M caused dose-and time-dependent accumulation of ubiquitinated proteins in PCa cells. The ubiquitinated proteins were noticeable as early as1h after treatment and sustained at high levels up to9h, then dropped down following18h of exposure in PC3and DU145cells.
二、Mar disrupts ER-associated degradation (ERAD) trigerring ER stress and apoptosis
1. Mar-mediated proteasome Inhibition inhibits ERAD resulting in ER stress.
Mar dramatically increased SPCΔ4levels in PCa cells transfected with SPCA4, a specific substrate of ERAD. Similar to the observation in MG132treatment, whereas neither Mar nor MG132affected SPCwt level. Effect of Mar on ERAD was also monitored by the localization of a green fluorescent protein in PC3cells transfected with pGFP-CFTRΔF508, another substrate of ERAD. In the absence of Mar, GFP in transfected cells was detected in both cytoplasm and ER, while Mar-treated cells displays primarily green fluorescence in the ER. Similar observations were shown in cells treated with MG132. These results indicated that Marchantin M exerted anti-proteasome activity and prevented ERAD. The transmission electron microscopy (TEM) revealed that the ER was moderately dilated in cells exposed to5μM Mar. However, the vacuolation of ER during high dose Mar treatment was observed in PC3and DU145cells. The expressin of the GRP78increased markedly following a short exposure to Marchantin M, and was sustained at high levels throughout the duration of treatment in three PCa cell lines. PERK and eIF2a phosphorylation was up-regulated in response to Mar exposure in three PCa cell lines, however their total protein level was not affected by Mar. To further investigate the effects of Marchantin M on the ER stress, four important ER stress response transducers XBP1, ATF6, ATF4and its downstream ATF3were also examined in Marchantin M-treated cells. The spliced form of XBP1mRNA increased in PC3cells exposed to Mar as early as1h, then decreased with longer treatment. However, the activated XBP1sustained for24h in DU145and LNCaP. Real time PCR analysis revealed that the activating transcription factor4(ATF4) and ATF3mRNA levels were largely increased by Mar and sustained up to48h during treatment, and the levels of activating transcription factor6(ATF6) was slightly increased in Mar-treated cells. The above data indicated that inhibition of proteasome by Mar disrupted EARD and resulted in prolonged ER stress.
2. Mar induced apoptosis in PCa cells.
Mar inhibitis cell viability and promotes apoptosis in PCa cells in a dose-dependent fashion. However, the apoptotic rate was much higher in hormone-dependent prostate cancer LNCaP than that of in hormone-independent prostate cancer PC3and DU145. Western blotting analysis displayed that Mar activated ER specific caspase-4resulting in the induction of caspase3cleavage to proteolytic fragment in PC3cells.
3. caspase-independent cell death involes in Mar-induced cell death in HRPC
To investigate the role of apoptosis in Mar-induced cell death in PCa cells, z-VAD-fmk, a pan inhibitor of caspase was pretreated in presence and absence of Mar. The data showed z-VAD-fmk dramatically rescued cell death in LNCaP cells, however, partial rescution was observed in hormone refractory prostate cancer PC3and DU145cells, which indicated caspase-dependent and independent cell death involes in Mar-induced cell death.
Part II Marchantin M inhibits AR function through up-regulating stress responsive gene of ATF3
Androgen receptor (AR), a nuclear receptor, dissociated from heat shock protein when it bind its ligand, which in turn promotes AR nuclear location and dimerization. Activated AR binds to AR response elements (AREs) and regulates gene transcription,such as PSA. Androgen and its receptor plays key role in maintaining development, proliferation and differentiation in prostate gland. Previous report displayed that AR promoted development from hormone-dependent prostate cancer to horomone-independent prostate cancer. Abnormal androgen signaling due to aberrant expression mutations, or dysregulation of the AR gene has been linked to prostate tumorigenesis, and progression of prostate cancer into advanced, castration-resistant disease.
Activating transcription factor3(ATF3) belongs to one of ATF/CREB family. ATF3bound to ATF/CREB element and regulates gene expression by its bZIP domain. ATF3was dramatically induced to maintain cellular homeostasis when cells were exposed to genetic toxins, oxidative stress and ER stress. However, ATF3mediated apoptosis during lasted and overloaded stress.
Our data of Part I showed that marchantin M mainly induced apoptosis in LNCaP cell accompanying ATF3upregulation. Previous study disclosed forced expression of ATF3disrupting AR function. So we investigated whether upregulation of ATF3involved in inhibiting AR function and triggering apoptosis in LNCaP cell.
一、Mar M raised ATF3expression by ER stress signal of PERK/eIF2α/ATF4
1. Mar up-regulated ATF3expression by transcriptional activation.
Mar significantly increased ATF3promoter activity and promote its transcription in LNCaP cells. The MatInspector software was used to search for the regions of conserved transcription factor-binding site within the-1850to+34regions. The ATF3gene promoter (pATF3-1850/+34) contained multiple potential transcription factor-binding sites including ATF (-22/-18). To investigate which regions Mar affects transcriptional regulation of the ATF3gene, promoter activity was measured using five serial deletion constructs, the point mutation and deletion mutation constructs. Our data demonstrated that ATF binding sites was crucial to the ATF3transcription activation.
2. ER stress-mediated PERK/eIF2a/ATF4involved in Mar-induced ATF3expression.
Inhibition of PERK activation by dominant negative or ATF4expression by targeting siRNA dramatically decreased ATF3expression.
二、Mar induced apoptosis by up-regulation of ATF3in LNCaP cell
1. The inhibition of ATF3expression promoted cell survival. Knocking-down ATF3expression by siRNA significantly decreased apoptosis.
2. Mar-induced ATF3triggered apoptosis in LNCaP cell in a caspase-dependent manner. Depletion of ATF3expression suppressed activation of caspase3resulting in decreasing cleavage of PARP, a substrate of active caspase3.
The above results showed that the Mar promoted ATF3expression and induced canonical apoptosis in LNCaP cell.
三、ATF3inhibited AR function through protein interaction and promoted apoptosis in LNCaP
1. Mar significantly raised ATF3protein level in hormone-dependent prostate cancer.
Mar improved ATF3transcription in three prostate cacer cells. However the protein level of ATF3in hormone-dependent LNCaP cells was much higher than in hormone-independent prostate cancer cells PC3and DU145.
2. ATF3interacted with AR and inhibited its function
Co-precipitation assay showed that ATF3interacted with AR in LNCaP cells. In LNCaP cells, overexpression of ATF3significantly decreased PSA promoter activity and its mRNA levels. Other AR targeting genes, such as TMPRSS23and NKX3.1, were also significantly decreased. However, the genes expression restored when depleted ATF3by siRNA. pression. When AR, PSA promoter and different doses of ATF3expression vector were transfected into PC3cells, PSA promoter activity decreased with increasing amount of ATF3expression. The above results indicated that Mar-induced ATF3promotes apoptosis by interacting with AR and inhibiting its function in LNCaP cells.
Part III Marchantin M induces autophagic cell death in prostate cancers
Inactivation of caspase by z-VAD-fmk partially rescued marchantin-induced cell death in hormone refractory prostate cancer, which suggests that caspase-independent mechanisms may also contribute to its cytotoxic effect on PCa cells. Besides triggering endoplasmic reticulum stress (ER stress) and promoting apoptosis, proteasome inhibition also induces autophagy.
Autophagy is a catabolic pathway to maintain cellular homeostasis by eliminating injured or aging organelles and unwanted proteins, and mediating turnover of long-lived proteins. In addition to UPS, autophagy is another protein quality control system in eukaryotic cells. When cells suffers extra or intra-cellular stress, the level of autophagy could be dramatically stimulated as a cytoprotective response resulting in adaptation and survival. The hydrolytic products by enzymes of lysosome can be reused by cells. UPS and autophagy are considered the two major routes of protein degradation in eukaryotic cells and their mutual-exclusiveness and inter-dependence.Uusually proteasome inhibition activates autophagy. However, dysregulated or excessive autophagy could cause autophagic cell death, the type II programmed cell death. Several chemotherapeutic agents in mammalian cells have been shown to induce autophagic cell death.
一、 Proteasome inhibition paralleled with autophagy induction
1. Mar inhibited proteasome activities accompanying with ER stress and autophagy
Mar suppressed proteasome activities at1h in Mar-treated cells,consequrently, GRP78and LC3BII increased at2h after Mar treatment, which suggested that Mar triggered ER stress and autophagy by proteasome inhibition.
2. Mar dramatically induced autophagy in HRPC
Mar rapidly up-regulated autophagy levels in treated cells, as judged by the accumulation of autophagy marker protein LC3B and the accumulation of lipidated LC3BⅡ in a concentration-dependent manner. Electron microscopy of Marchantin M-treated PC3cells showed the formation of double-or multi-membranes engulfing high electron-density substances, evidenced as autohagosomes or autolysosomes. Marchantin M induced autophagy was also judged by punctate dots of a green fluorescent protein tagged form of LC3B (GFP-LC3B). The cells transfected with GFP-LC3B expression plasmid showed diffuse green fluorescence, while Marchantin M treatment caused significantly punctated green fluorescence in cytosol. The similar results were observed in GFP-LC3ransfected U87cells.3. Mar promoted autophagic flux in HRPC
Data of gene chip and qRT-PCR demonstated that Mar only dramatically induced LC3B expression, and other ATG genes shwoed insignificant difference. Western blotting displayed Mar not only induced LC3B expression but promotes turnover of LC3BⅠ. As increased LC3BⅠ to LC3BⅡ conversion could occur when autophagy was either stimulated or blocked. To further explore if autophagic flux can be induced by Marchantin M, we chose chloroquine (CQ), an agent that prevents the formation of autolysosomes by inhibiting autophagosome-lysosome fusion and LC3BⅡ degradation. The result showed that Marchantin M-stimulated processing of LC3B was enhanced in the presence of CQ, whereas no detectable change was observed in cells exposed to CQ alone, suggesting that Marchantin M has the potential to promote autophagosome formation. Thus, Marchantin M was effective in inducing a prolonged autophagy.
二、Mar induced autophagic cell death in hormone-independent prostate cancer cells
1. Pharmacological blockade of autophagy rescued Mar-mediated cell death
Mar treatment led to the accumulation of LC3BⅡ, whereas this effect was significantly blocked in the presence of3-MA, and accompanied with an increase in cell viability and reduction in cell death. Similarly, Mar-mediated induction of LC3BⅡ was enhanced in combination with E-64D/Pepstatin A, another autophagy inhibitor that prevents lysosomal enzyme activation, and cell proliferation was significantly restored and cell death was markedly decreased in co-treatment.
2. knock-down of LC3B, Atg5and Atg7attenuated Marchantin M-mediated cell death Knockdown of endogenous levels of Atg5by Atg5-targeting siRNA dramatically increased cell viability and blocked cell death after24and48h transfection followed by Mar treatment. Mar-induced LC3BII levels were markedly suppressed when Atg5was down-regulated by siRNA. Similar to the observations of Atg5, reduction of LC3B or Atg7by siRNA significantly alleviated cytotoxic activity of Mar. Taken together, our data clearly emonstrated that Mar activated an autophagic flux and promoted autophagy-dependent cell death.
3. Combined inhibition of caspases and autophagy almost completely rescued Mar-induced cell death
Caspase inhibitors only partially reversed to Mar-induced cell death in hormone-independent prostate cancer cells. Depletion of Atg5expression by siRNA in conjunction with the caspase inhibitor almost completely blocked the cell death induced by mar, which indicated both autophagic cell death and caspase-dependent apoptosis involved in cytotoxicity to hormone-independent prostate cancer cells.
三、PERK/PERK/eIF2α和PI3K/Akt/mTOR involved in Mar-mediated autophagy
1. Mar inhibited PI3K/Akt/mTOR signal by preventing Aktl transcription resulting in autophagy activation.
Western blotting results showed that Mar decreased the phosphorylation level of PDK1, Akt and total Akt protein resulting in reducing phospho-mTOR and phospho-p70S6K. The attenuation of AKT by Mar was further confirmed at mRNA level in PC3cells and the expression of AKT3decreased much lower than that of AKT1/2. Forced expression of AKT-wt was unable to increase the phosphorylation level of GSK3β, a downstream target of AKT, as well as the cell viability exposed to Mar. Unlike to the AKT-wt, PC3cells transfected with AKT-myr showed significant increase in phospho-GSK3β level and cell viability, accompanied with a decrease in the LC3BII. The results showed that Mar inhibited expression and activation of Akt leading to mTOR inactivation and inducing autophagy.
2. Mar-induced ER stress signaling promotes autophagy
Blockade of JNK/c-Jun signal with SP600125hardly affected Mar-induced LC3BII expression and cell death. To explore a link between PERK/eIF2a signaling and autophagic activation in response to proteasome inhibition by Mar, we performed transient transfection with dominant negative PERK (PERK-DN) expression plasmid into cells to impair the function of PERK. The results displayed that Mar-induced eIF2a phosphorylation was blunted by inactivation of PERK. More importantly, co-treatment of cells with Mar and PERK-DN significantly caused the blocking of LC3BII accumulation and partial restoration of viable cells as well as decreased cell death. These results indicate that endoplasmic reticulum stress signal PERK/eIF2a was involved in Mar-mediated autophagy and cell death.
Part IV Conclusions and Innovation
一、Conclusions
1. Mar inhibited proteasome activities in vitro and established PCa cell lines, which indicated Mar was a novel revesible inhibitor of proteasome.
2. Mar disrupted endoplasmic reticulum associated degradation pathway by inhibiting proteasome activity resulting in ER stress, by which promoted cell death in PCa cells.
3. Mar up-regluated ATF3, a stress mediator, in hormone-dependent prostate cancer, which in turn inhibits AR function and promoted apoptosis in LNCaP cells.
4. Mar M indces caspase-dependent and independent cell death in androgen-independent prostate cancer cells by induction of ER stress and autophagy.
二、Innovation and defects
1. We Firstly reported Mar a macrocyclic bisbibenzyls targeted the proteasome activities and showed its inhibitory effect in a reversible manner. Since irreversible inhibitors of proteasome usually cause normal cell apoptosis and death, reversible inhibitors are more valuable in drug discovery. Our data suggests that Mar may represent a promising compound in the future development to treat PCa.
2. We firstly reported Mar prevented AR function by up-regulation of stress mediator ATF3and promoted apoptosis in LNCaP. Since AR in LNCaP cells was mutant, the characteristics of Mar will be beneficial for the treatment of prostate cancer expression constitutive activation of the AR.. However, the effect of Mar on wild-type AR needed to be investigated.
3. Mar triggered autophagic cell death in hormone-independent prostate cancer. Since most chemotherapy agents such as paclitaxel induced autophagic protection in tumor cells, which in turn weakened its anti-tumor effect. Moreover hormone-independent PCa cells showed more potential to anti-apoptosis. However the IC50values of Mar are similar between hormone-dependent and hormone-independent PCa cells and Mar induced autophagic cell death in hormone-independent PCa cells, which conferred Mar a promising anticancer drugs. But animal pharmacodynamics of the compounds needed to be testified.
前列腺癌是激素依赖性肿瘤,因此内分泌治疗是治疗前列腺癌的首选标准方案。然而绝大多数前列腺癌患者经内分泌治疗1-2年后易发展为激素难治性前列腺癌(Hormone Refractory Prostate Cancer,HRPC),对雄激素撤除不敏感,抵抗凋亡并伴随侵袭转移,预后差,死亡率高,目前临床尚无有效的治疗方法。由于HRPC的演变是复杂的多因素、多信号途径参与的过程,其机制尚不完全清楚,因此寻找有效的治疗靶点及机制,产生多种途径的协同效应,是治疗前列腺癌的有效策略之一。
第一部分地钱素M抑制蛋白酶体的活性,诱导细胞内质网应激和凋亡
泛素-蛋白酶体系统(Ubiquitin-proteasome system, UPS)是真核生物细胞内重要的蛋白质质量控制体系之一。UPS通过将小分子蛋白泛素与靶蛋白的赖氨酸残基共价结合,从而将其泛素化。泛素标记的蛋白被蛋白酶体特异性地识别并迅速降解。UPS通常降解受损变性蛋白、短寿命的调控蛋白等,对于细胞周期运转、凋亡与分化、信号转导等生理过程起着重要的调控作用。恶性肿瘤细胞内蛋白代谢紊乱,蛋白酶体活性升高,UPS调节失衡,使得肿瘤细胞更加依赖UPS,以维持细胞内稳态。因此,肿瘤细胞对蛋白酶体抑制剂更加敏感,蛋白酶体也成为肿瘤治疗的新靶点,开发针对UPS的抑制剂在肿瘤治疗中具有广阔的前景,而天然化合物以其独特的结构、多样的生物学活性更是备受关注。
双联苄(Bisbibenzyls)是苔藓植物中分离得到的一类大环多酚类化合物,因联苄之间的连接方式不同、取代基不同而具有结构新颖、多样的特点,具有抗真菌、抗炎、抗肿瘤等多种生物活性。本课题组前期研究发现双联苄类化合物地钱素M(Marchantin M,Mar)显著抑制前列腺癌细胞的增殖、促进其凋亡。由于地钱素M的结构与茶多酚的功能区域相似,而茶多酚已被证明是蛋白酶体的不可逆抑制剂。我们推测地钱素M可能具有抑制蛋白酶体活性的功能。为此,我们研究了地钱素M对重组纯化的20S蛋白酶体、前列腺癌细胞内蛋白酶体活性的影响,并对其生物学效应进行了分析,取得了以下研究结果：
一、地钱素M显著抑制蛋白酶体活性,是新的可逆性蛋白酶体抑制剂
1.地钱素M显著抑制蛋白酶体胰凝乳样蛋白酶(ChT-L)和肽基谷氨酰多肽解酶(PGPH)活性。Mar显著抑制重组纯化的20S蛋白酶体中ChT-L和PGPH活性,呈剂量依赖性。其半数抑制率分别为6.99和5.33μM。但不影响胰蛋白酶样活性。细胞实验表明,地钱素M对激素非依赖性前列腺癌PC3,DU145细胞和激素依赖性前列腺癌LNCaP细胞内26S蛋白酶体显示同样的抑制作用,且呈时间和剂量依赖性。表达差异谱基因芯片数据显示,地钱素M并不影响UPS途径中各组分的基因表达。
2.地钱素M是新的可逆性蛋白酶体抑制剂。通过Autodock vina软件分析发现,地钱素M能与蛋白酶体的β1(PGPH)和β5(ChT-L)亚基结合。二者主要依靠氢键、疏水键等非共价键相互作用,其自由能分别为：-7.4kcal/mol和-6.5kcal/mol。林-贝氏双倒数作图法表明,地钱素M对蛋白酶体的抑制呈现较典型的非竞争性抑制的方式。上述结果表明,地钱素M是一种新的蛋白酶体可逆的抑制剂。
3.地钱素M抑制蛋白酶体活性,导致前列腺癌细胞内多泛素化蛋白累积,且呈时间、剂量依赖性。
二、地钱素M抑制蛋白酶体活性,导致内质网相关蛋白降解途径(ERAD)受阻,诱导内质网应激和细胞凋亡
1.地钱素M介导的蛋白酶体抑制,抑制ERAD,诱导内质网应激。地钱素M处理前列腺癌细胞后,导致经ERAD降解的蛋白如突变蛋白CFTRAF508、SPCA4在内质网堆积；电镜下观察到内质网肿胀,激光共聚焦结果显示高浓度的地钱素M可引起内质网空泡化；Western blotting结果表明,内质网伴侣蛋白GRP78表达显著上调、PERK及其下游eIF2α磷酸化水平升高；定量PCR分析显示应激分子ATF6.ATF4及其靶基因ATF3表达显著增加；IRE1激活并剪接XBP1的mRNA,激活XBP1。上述结果表明,地钱素M抑制蛋白酶体活性导致内质网内未折叠或错误折叠的蛋白累积,诱导内质网应激。
2.地钱素M诱导前列腺癌细胞凋亡。地钱素M呈剂量依赖性抑制激素依赖、激素非依赖性前列腺癌细胞凋亡,且使激素依赖性细胞更易凋亡。Western blotting结果表明,地钱素M可激活内质网特异caspase4,从而激活caspase3,诱导凋亡。
3.Caspase非依赖途径参与地钱素M诱导的细胞凋亡。在激素依赖的前列腺癌LNCaP细胞中,Caspase抑制剂z-VAD-fmk逆转绝大多数地钱素M诱导的细胞死亡,而在激素非依赖的前列腺癌PC3和DU145细胞中,z-VAD-fmk只能部分部分逆转地钱素M介导的细胞死亡。上述结果表明,地钱素M主要通过caspase依赖的途径诱导激素依赖性前列腺癌细胞凋亡,而通过caspase依赖和非依赖机制诱导激素非依赖性前列腺癌细胞死亡。
第二部分地钱素M上调应激基因ATF3的表达而抑制激素依赖性前列腺癌细胞中AR的功能
雄激素受体(Androgen receptor,AR)为核受体型转录因子。当雄激素(如睾酮、二氢睾酮)与AR结合后,引起AR构象改变,使AR与热休克蛋白解离、活化而转移至核内,并形成同源二聚体与DNA分子中雄激素受体应答元件(AREs)结合,诱导下游靶基因如前列腺特异性抗原(prostate specific antigen,PSA)等基因的转录表达,促进细胞生长和存活。因此,AR信号通路不仅在维持前列腺的正常发育、功能发挥中起主要作用,也是激素依赖性前列腺癌增殖的关键途径,而且在激素非依赖性转化过程中起决定性作用,并在约1/3的HRPC中持续性高表达。AR基因扩增、突变、过表达以及AR激素非依赖性激活,均可引起AR功能的异常活化,是前列腺癌内分泌治疗失败、侵袭转移、抵抗凋亡等恶性表型的重要机制之一。因此,有效抑制前列腺癌中AR的表达或和功能一直是倍受关注的研究课题。
ATF3(activating transcription factor3)作为转录因子ATF/CREB家族成员,通过其蛋白碱性区的亮氨酸拉链结合到特异DNA的ATF/CREB顺式作用元件上,调控基因表达。当DNA损伤、毒素暴露、氧化应激以及内质网应激时,ATF3可被迅速诱导表达,以维持细胞在应激条件下的基因组完整性和内稳态。然而,在持续或过强的应激状态下,ATF3可介导细胞凋亡。由于ATF3蛋白中的亮氨酸拉链可介导ATF3与多种蛋白相互作用。本课题第一部分研究结果显示,地钱素M诱导内质网应激,显著增加ATF3表达,且主要以caspase依赖的凋亡形式诱导激素依赖性前列腺癌细胞LNCaP死亡。由于AR是LNCaP细胞增殖存活的关键调控因子,我们研究了ATF3高表达对AR表达和功能的影响以及与LNCaP凋亡的关系,结果如下：
一、地钱素M通过内质网应激信号PERK/eIF2α/ATF4上调ATF3的表达
1.地钱素M在转录水平上调ATF3的表达。地钱素M显著增加前列腺癌细胞中ATF3启动子活性,促进其转录。利用MatInspector软件分析ATF3启动子区转录因子的结合位点,发现-22/-18区存在ATF的结合位点；通过构建ATF3启动子及其系列截短体和ATF结合区的点突变和缺失突变的报告基因载体。双荧光素酶检测结果表明,ATF结合位点对地钱素M诱导的ATF3转录激活至关重要。
2.地钱素M诱导的ATF3表达上调主要受内质网应激信号PERK/eIF2α/ATF4的调控。通过显性负相关表达载体抑制PERK的活性或siRNA下调ATF4的表达均能显著下调ATF3的表达,同时促进细胞存活。
二、地钱素M诱导的ATF3高表达促进LNCaP细胞凋亡
1.抑制ATF3的表达促进细胞存活。利用显性负相关或siRNA技术抑制ATF3的表达,细胞增殖实验结果显示LNCaP细胞的存活率显著提高；AnnexinV-FITC/PI双染结果表明,下调ATF3后,凋亡细胞显著减少。
2.ATF3通过caspase途径诱导细胞凋亡。Western-blotting结果显示,降低ATF3的表达,可抑制caspase3的活化,使caspase3的底物PARP剪切减少。上述结果表明,地钱素M上调ATF3的表达促进LNCaP经caspase依赖的途径凋亡。
三、高表达的ATF3通过蛋白间相互作用抑制AR的功能、促进LNCaP细胞凋亡
1.地钱素M显著上调ATF3蛋白水平。Western-blotting结果显示,地钱素M显著提高ATF3的蛋白表达,而且在激素依赖性前列腺癌细胞LNCaP中的诱导作用远远高于激素非依赖性前列腺癌细胞PC3和DU145。
2.ATF3与AR相互作用抑制其功能。免疫共沉淀结果显示,地钱素M诱导的ATF3与AR结合。在LNCaP细胞中过表达ATF3显著下调AR的靶基因PSA的启动子活性及其mRNA水平,其它AR调控的靶基因,如TMPRSS23和NKX3.1等表达均明显下调。利用siRNA干扰ATF3后,上述AR靶基因都有不同程度的恢复表达。当AR缺失的PC3细胞共转染AR,PSA启动子和不同剂量的ATF3表达载体后,PSA启动子活性随ATF3表达量的增加而降低。以上结果说明地钱素M诱导的ATF3通过与AR结合而抑制其功能,从而诱导LNCaP细胞凋亡。
第三部分地钱素M诱导激素非依赖性前列腺癌细胞自噬性死亡
在激素非依赖性前列腺癌细胞PC3和DU145中,阻断caspase依赖的细胞凋亡,只能部分逆转地钱素M诱导的细胞死亡,提示caspase非依赖的细胞死亡机制参与地钱素M诱导的细胞死亡过程。
已有研究表明,抑制蛋白酶体活性不仅能够促进肿瘤细胞凋亡,还可通过多种机制诱导细胞自噬(autophagy)。自噬是真核细胞内除UPS外,另外一种蛋白质质量控制系统,主要通过自噬泡包裹细胞胞液的内容物,包括衰老或受损的亚细胞器如线粒体、长寿命蛋白、入侵的微生物等运送至溶酶体降解,降解产物可以被细胞再次利用。当细胞面临各种刺激、应激时,自噬可被诱导增强,可通过出现的自噬泡、自噬相关基因的表达等方法进行检测。自噬对肿瘤细胞具有双重作用,在肿瘤发生的不同阶段,自噬所起的作用不同。在正常条件下,基础性自噬可清除细胞内可能产生毒性作用的受损成分,抑制细胞的恶性转化,预防肿瘤发生。当肿瘤细胞面临乏氧、营养缺乏以及化疗药物时,自噬的激活会提供能量、营养物质等而对肿瘤细胞起到保护作用,有利于细胞存活。然而多种应激而产生持续、过度的自噬时,也会导致细胞死亡,称为自噬性细胞死亡(autophagic cell death),即Ⅱ型程序性细胞死亡。地钱素M处理的前列腺癌细胞,不仅使自噬的标志性蛋白LC3的表达增加,而且促进LC3I向LC3Ⅱ转变,表明地钱素M能诱导前列腺癌细胞产生自噬。因此,我们研究了地钱素M诱导的自噬在前列腺癌细胞死亡过程中的作用,并对其信号通路进行了探讨,取得了以下研究成果：
一、地钱素M抑制蛋白酶体活性,几乎同时诱导自噬的发生
1.地钱素M抑制蛋白酶体活性伴随着内质网应激和细胞自噬。时相分析结果显示,地钱素M处理细胞1h时,蛋白酶体活性显著下降,而在处理2h后内质网伴侣蛋白GRP78的表达显著上调、自噬标志性蛋白LC3Ⅱ的表达开始上调并持续增强至48h。此结果表明,地钱素M抑制蛋白酶体活性后,随即引起内质网应激和细胞自噬。
2.地钱素M显著诱导细胞产生自噬。LC3Ⅱ的表达升高提示该化合物可使细胞产生自噬。透射电镜的结果明确显示,地钱素M使激素非依赖性前列腺癌细胞中自噬泡和自噬溶酶体明显增多,为自噬提供直接证据。激素非依赖性前列腺癌细胞瞬时转染GFP-LC3表达载体或利用GFP-LC3稳转的U87细胞株,均可观察到地钱素M极显著地诱导LC3聚集、产生聚集荧光斑点。
3.地钱素M诱导自噬流量的增加。基因芯片和定量PCR结果表明,地钱素M在转录水平诱导LC3基因表达升高,而其它自噬相关基因如Atg5、Atg7、Beclin1等表达几乎没有变化。Western blotting结果显示地钱素M不仅上调LC3的蛋白水平,而且促进LC3I向LC3Ⅱ转变,并引起自噬底物p62下降。利用氯喹阻断自噬泡和溶酶体融合进一步增加地钱素M诱导的LC3Ⅱ的累积,而利用自噬抑制剂3-MA则显著抑制地钱素M诱导的LC3荧光斑点的聚集。这些结果表明地钱素M抑制蛋白酶体活性后,迅速诱导内质网应激和细胞自噬,显著增加自噬流量。
二、地钱素M诱导激素非依赖性前列腺癌细胞自噬性死亡
1.自噬抑制剂逆转地钱素M诱导的细胞死亡。自噬抑制剂3-MA显著下调地钱素M诱导的LC3Ⅱ的表达,同时促进细胞存活,降低地钱素M介导的细胞死亡。利用E-64D/Pepstatin A抑制溶酶体酶的活性,使地钱素M诱导的LC3Ⅱ的表达略有增加,但依然显著增加细胞的存活,逆转地钱素M诱导的细胞死亡。
2.抑制自噬相关基因逆转地钱素M诱导的细胞死亡。利用siRNA特异性的降低Atg5、Atg7、LC3的表达,均可显著抑制LC3Ⅱ的表达或增加p62的表达而抑制自噬,同时显著逆转地钱素M诱导的细胞死亡,增加细胞存活率。然而干扰Beclinl后并不影响地钱素M介导的细胞死亡。上述结果表明,地钱素M诱导的自噬为自噬性死亡。
3.抑制自噬和caspase凋亡途径完全逆转地钱素M诱导的细胞死亡。由于caspase抑制剂只部分逆转地钱素M诱导的激素非依赖性前列腺癌细胞凋亡,采用siRNA下调Atg5的表达抑制自噬、同时联用caspase抑制剂,几乎完全逆转地钱素M诱导的激素非依赖性前列腺癌细胞死亡。因此,白噬性细胞死亡和caspase依赖的细胞凋亡并存于地钱素M的细胞毒作用。
三、地钱素M通过内质网应激信号PERK/eIF2α和PI3K/Akt/mTOR途径诱导自噬
1.地钱素M通过下调Akt1的表达抑制PI3K/Akt/mTOR途径诱导自噬。Westernblotting结果显示,地钱素M首先下调phospho-PDK1、phospho-Akt以及总Akt蛋白的表达水平,随后降低phospho-mTOR和phospho-p70S6K的水平,表明地钱素M通过抑制PI3K/Akt/mTOR途径而诱导自噬。定量PCR结果显示,与Akt2、Akt3相比,地钱素M极显著地抑制Akt1的mRNA水平。持续活化的Aktl表达载体能够显著逆转地钱素M诱导的LC3Ⅱ表达及细胞死亡,而野生型Aktl无此作用。此结果表明,地钱素M通过下调Akt的表达、抑制PI3K的活性,使Akt磷酸化水平下降,从而使mTOR失活而诱导自噬。
2.地钱素M诱导的内质网应激信号促进白噬。利用JNK抑制剂阻断JNK/c-Jun信号并不影响地钱素M诱导LC3Ⅱ表达及细胞死亡,而转染显性负相关的PERK表达载体或siRNA抑制PERK,阻断内质网应激信号PERK/eIF2α,地钱素M诱导的LC3Ⅱ表达显著下调、并部分逆转其诱导的细胞死亡。上述结果表明,内质网应激信号PERK/eIF2α参与了地钱素M诱导的自噬及细胞死亡过程。
第四部分结论及创新点
一、结论
1.双联苄化合物地钱素M抑制蛋白酶体活性,是一新型可逆性蛋白酶体抑制剂。
2.地钱素M通过抑制蛋白酶体活性、阻断内质网相关的蛋白降解途径而诱导内质网应激,促进前列腺癌细胞死亡。
3.地钱素M通过诱导内质网应激、促进应激转录因子ATF3的高表达；而高表达的ATF3通过与AR发生蛋白间相互作用抑制AR功能,从而诱导激素依赖性前列腺癌细胞主要发生细胞凋亡。
4.地钱素M通过诱导内质网应激和细胞自噬,促进激素非依赖性前列腺癌细胞发生caspase依赖的凋亡和自噬性细胞死亡
二、创新点与不足之处
1.首次报道双联苄化合物地钱素M靶向蛋白酶体、是一新型的可逆性蛋白酶体抑制剂。由于不可逆性蛋白酶体抑制剂易产生较大毒性,因此地钱素M的作用特点,使其成为很有研究价值的天然产物。
2.首次报道地钱素M可通过应激蛋白ATF3而抑制AR的功能,进而促进细胞凋亡。由于LNCaP细胞表达的是突变型AR,地钱素M的这种抑制作用将有益于AR持续活化的前列腺癌的治疗。但地钱素M对野生型AR的作用有待于进一步研究。
3.首次报道地钱素M可诱导激素非依赖性前列腺癌自噬性细胞死亡。由于大多数化疗药物如紫杉醇等可引起肿瘤细胞产生自噬性保护机制,削弱其抗肿瘤效果,而且激素非依赖性前列腺癌细胞比激素依赖性细胞抗凋亡能力强,对药物敏感性差。地钱素M对激素依赖、非依赖前列腺癌细胞的IC50值相近,且可引起自噬性细胞死亡,这些特点使地钱素M是很有潜力的抗肿瘤药物。但该化合物的动物药效学实验尚需进行。
Prostate cancer is the most common nonskin cancer in old men in the Western world. While improved early detection significantly decreased mortality, PCa still remains the second leading cause of cancer-related death in Western men. Although the incidence of prostate cancer was lower than the Western countries', in recent years confirmed PCa, especially for advanced or metastatic PCa, represents an obviously rising trend in China with the aging and the diet habit changing.
PCa is initially androgen-dependent with complicated pathogenesis. In addition to genetic factors, the incidence of prostate cancer is related with high levels of androgens, race, diet and lifestyle. PCa depends largely on androgen receptor (AR) signaling for growth and maintenance. Androgen deprivation therapy has become the standard first-line treatment for advanced hormone naive PCa. Unfortunately, the development of hormone-refractory prostate cancer (HRPC) has been observed to occur within a few years after hormonal deprivation therapy and is associated with poor prognosis, high apoptosis-resistance and high mortality. There are few efficacious treatment options available for curing, or even improving the survival and quality of life in patients with HRPC. Complicated factors and signals involves in the development of HRPC. Nevertheless, the underlie mechanism of PCa is still remains elusive. Therefore, novel strategies targeting the molecular basis of PCa progression and producing synergies of different signals are highly desirable.
Part I Marchantin M inhibits the activities of the proteasome and triggering ER stress and apoptosis
Ubiquitin-proteasome system (UPS) is one of protein quality control system in eukaryotic cells. UPS works mainly through ubiquitination of the target protein and degradation of abnormal proteins and short-lived protein by proteasome. UPS plays an essential role in cell proliferation, cell cycle regulation, immune response, signal transduction and cell death. Recent studies have validated that proteasome activities in malignant tumors are much higher than that in non-malignant cells, conferring tumor cells more dependent on the UPS to maintain cellular homeostasis. Therefore, tumor cells are more sensitive to UPS inhibitors. UPS has gradually become a new target for cancer therapy, and Reagents targeting at proteasome are expected to become promising anticancer drugs. Natural products have gained much attention in their unique structures and diverse bioactivities.
Macrocyclic bisbibenzyls are a series of phenolic natural products isolated from liverwort, which exert a variety of biological activities, including antifung, antioxidation and cytotoxicity. Our previous research demonstrates its inhibitory effect on PCa cells via activation of caspase pathway.
Based on the nature of its structure, Marchantin M (Mar) was somewhat similar to tea polyphenols, bearing with phenol groups which may act as tyrosine mimic binding to β5subunit of proteasome. We speculate that Marchantin M may be a novel proteasome inhibitor. Therefore, we discuss the inhibitory effect of Marchantin M on proteasome in prostate cancer cells.
一、Mar is a novel reversible inhibitors targeting proteasome activities
1. Mar dramatically inhibits ChT-L and PGPH activities of proteasome.
Mar inhibits ChT-L and PGPH activities of proteasome of20S proteasome in a dose-dependent manner in vitro. The IC50values for ChT-L and PGPH were6.99and5.33μM, respectively. However, Marchantin M hardly inhibited Try-L activity of the 20S proteasome. The Similar results were observed in Mar-treated PCa cells. Analysis of gene chip shows that Mar inhibits proteasome activities rather than suppresses expression of UPS components. The results indicate Mar is a novel inhibitor.
2. Mar is a reversible inhibitor of proteasome.
Since the ChT-L and PGPH activities were mediated by the β5and β1subunits of proteasome, we docked the Mar molecule into β5and β1subunits to investigate their interactions by autodock vina. The docking results revealed that Mar bound to P5and β1subunits at the active sites with a conformation suitable for proteasome inhibition. The docked free energy was-7.4kcal/mol and-6.5kcal/mol, respectively. The docking images also indicated that Marchantin M interacted with β1and β5subunits through hydrogen and hydrophobic interactions. Lineweaver-Burk double-reciprocal plots for the PGPH activity displayed characteristics of non-competitive inhibition, the Km value was determined to be48.52μM. The data clearly demonstrates that Mar is a novel reversible inhibitor of proteasome.
3. In accordance with proteasome inhibition, Marchantin M caused dose-and time-dependent accumulation of ubiquitinated proteins in PCa cells. The ubiquitinated proteins were noticeable as early as1h after treatment and sustained at high levels up to9h, then dropped down following18h of exposure in PC3and DU145cells.
二、Mar disrupts ER-associated degradation (ERAD) trigerring ER stress and apoptosis
1. Mar-mediated proteasome Inhibition inhibits ERAD resulting in ER stress.
Mar dramatically increased SPCΔ4levels in PCa cells transfected with SPCA4, a specific substrate of ERAD. Similar to the observation in MG132treatment, whereas neither Mar nor MG132affected SPCwt level. Effect of Mar on ERAD was also monitored by the localization of a green fluorescent protein in PC3cells transfected with pGFP-CFTRΔF508, another substrate of ERAD. In the absence of Mar, GFP in transfected cells was detected in both cytoplasm and ER, while Mar-treated cells displays primarily green fluorescence in the ER. Similar observations were shown in cells treated with MG132. These results indicated that Marchantin M exerted anti-proteasome activity and prevented ERAD. The transmission electron microscopy (TEM) revealed that the ER was moderately dilated in cells exposed to5μM Mar. However, the vacuolation of ER during high dose Mar treatment was observed in PC3and DU145cells. The expressin of the GRP78increased markedly following a short exposure to Marchantin M, and was sustained at high levels throughout the duration of treatment in three PCa cell lines. PERK and eIF2a phosphorylation was up-regulated in response to Mar exposure in three PCa cell lines, however their total protein level was not affected by Mar. To further investigate the effects of Marchantin M on the ER stress, four important ER stress response transducers XBP1, ATF6, ATF4and its downstream ATF3were also examined in Marchantin M-treated cells. The spliced form of XBP1mRNA increased in PC3cells exposed to Mar as early as1h, then decreased with longer treatment. However, the activated XBP1sustained for24h in DU145and LNCaP. Real time PCR analysis revealed that the activating transcription factor4(ATF4) and ATF3mRNA levels were largely increased by Mar and sustained up to48h during treatment, and the levels of activating transcription factor6(ATF6) was slightly increased in Mar-treated cells. The above data indicated that inhibition of proteasome by Mar disrupted EARD and resulted in prolonged ER stress.
2. Mar induced apoptosis in PCa cells.
Mar inhibitis cell viability and promotes apoptosis in PCa cells in a dose-dependent fashion. However, the apoptotic rate was much higher in hormone-dependent prostate cancer LNCaP than that of in hormone-independent prostate cancer PC3and DU145. Western blotting analysis displayed that Mar activated ER specific caspase-4resulting in the induction of caspase3cleavage to proteolytic fragment in PC3cells.
3. caspase-independent cell death involes in Mar-induced cell death in HRPC
To investigate the role of apoptosis in Mar-induced cell death in PCa cells, z-VAD-fmk, a pan inhibitor of caspase was pretreated in presence and absence of Mar. The data showed z-VAD-fmk dramatically rescued cell death in LNCaP cells, however, partial rescution was observed in hormone refractory prostate cancer PC3and DU145cells, which indicated caspase-dependent and independent cell death involes in Mar-induced cell death.
Part II Marchantin M inhibits AR function through up-regulating stress responsive gene of ATF3
Androgen receptor (AR), a nuclear receptor, dissociated from heat shock protein when it bind its ligand, which in turn promotes AR nuclear location and dimerization. Activated AR binds to AR response elements (AREs) and regulates gene transcription,such as PSA. Androgen and its receptor plays key role in maintaining development, proliferation and differentiation in prostate gland. Previous report displayed that AR promoted development from hormone-dependent prostate cancer to horomone-independent prostate cancer. Abnormal androgen signaling due to aberrant expression mutations, or dysregulation of the AR gene has been linked to prostate tumorigenesis, and progression of prostate cancer into advanced, castration-resistant disease.
Activating transcription factor3(ATF3) belongs to one of ATF/CREB family. ATF3bound to ATF/CREB element and regulates gene expression by its bZIP domain. ATF3was dramatically induced to maintain cellular homeostasis when cells were exposed to genetic toxins, oxidative stress and ER stress. However, ATF3mediated apoptosis during lasted and overloaded stress.
Our data of Part I showed that marchantin M mainly induced apoptosis in LNCaP cell accompanying ATF3upregulation. Previous study disclosed forced expression of ATF3disrupting AR function. So we investigated whether upregulation of ATF3involved in inhibiting AR function and triggering apoptosis in LNCaP cell.
一、Mar M raised ATF3expression by ER stress signal of PERK/eIF2α/ATF4
1. Mar up-regulated ATF3expression by transcriptional activation.
Mar significantly increased ATF3promoter activity and promote its transcription in LNCaP cells. The MatInspector software was used to search for the regions of conserved transcription factor-binding site within the-1850to+34regions. The ATF3gene promoter (pATF3-1850/+34) contained multiple potential transcription factor-binding sites including ATF (-22/-18). To investigate which regions Mar affects transcriptional regulation of the ATF3gene, promoter activity was measured using five serial deletion constructs, the point mutation and deletion mutation constructs. Our data demonstrated that ATF binding sites was crucial to the ATF3transcription activation.
2. ER stress-mediated PERK/eIF2a/ATF4involved in Mar-induced ATF3expression.
Inhibition of PERK activation by dominant negative or ATF4expression by targeting siRNA dramatically decreased ATF3expression.
二、Mar induced apoptosis by up-regulation of ATF3in LNCaP cell
1. The inhibition of ATF3expression promoted cell survival. Knocking-down ATF3expression by siRNA significantly decreased apoptosis.
2. Mar-induced ATF3triggered apoptosis in LNCaP cell in a caspase-dependent manner. Depletion of ATF3expression suppressed activation of caspase3resulting in decreasing cleavage of PARP, a substrate of active caspase3.
The above results showed that the Mar promoted ATF3expression and induced canonical apoptosis in LNCaP cell.
三、ATF3inhibited AR function through protein interaction and promoted apoptosis in LNCaP
1. Mar significantly raised ATF3protein level in hormone-dependent prostate cancer.
Mar improved ATF3transcription in three prostate cacer cells. However the protein level of ATF3in hormone-dependent LNCaP cells was much higher than in hormone-independent prostate cancer cells PC3and DU145.
2. ATF3interacted with AR and inhibited its function
Co-precipitation assay showed that ATF3interacted with AR in LNCaP cells. In LNCaP cells, overexpression of ATF3significantly decreased PSA promoter activity and its mRNA levels. Other AR targeting genes, such as TMPRSS23and NKX3.1, were also significantly decreased. However, the genes expression restored when depleted ATF3by siRNA. pression. When AR, PSA promoter and different doses of ATF3expression vector were transfected into PC3cells, PSA promoter activity decreased with increasing amount of ATF3expression. The above results indicated that Mar-induced ATF3promotes apoptosis by interacting with AR and inhibiting its function in LNCaP cells.
Part III Marchantin M induces autophagic cell death in prostate cancers
Inactivation of caspase by z-VAD-fmk partially rescued marchantin-induced cell death in hormone refractory prostate cancer, which suggests that caspase-independent mechanisms may also contribute to its cytotoxic effect on PCa cells. Besides triggering endoplasmic reticulum stress (ER stress) and promoting apoptosis, proteasome inhibition also induces autophagy.
Autophagy is a catabolic pathway to maintain cellular homeostasis by eliminating injured or aging organelles and unwanted proteins, and mediating turnover of long-lived proteins. In addition to UPS, autophagy is another protein quality control system in eukaryotic cells. When cells suffers extra or intra-cellular stress, the level of autophagy could be dramatically stimulated as a cytoprotective response resulting in adaptation and survival. The hydrolytic products by enzymes of lysosome can be reused by cells. UPS and autophagy are considered the two major routes of protein degradation in eukaryotic cells and their mutual-exclusiveness and inter-dependence.Uusually proteasome inhibition activates autophagy. However, dysregulated or excessive autophagy could cause autophagic cell death, the type II programmed cell death. Several chemotherapeutic agents in mammalian cells have been shown to induce autophagic cell death.
一、 Proteasome inhibition paralleled with autophagy induction
1. Mar inhibited proteasome activities accompanying with ER stress and autophagy
Mar suppressed proteasome activities at1h in Mar-treated cells,consequrently, GRP78and LC3BII increased at2h after Mar treatment, which suggested that Mar triggered ER stress and autophagy by proteasome inhibition.
2. Mar dramatically induced autophagy in HRPC
Mar rapidly up-regulated autophagy levels in treated cells, as judged by the accumulation of autophagy marker protein LC3B and the accumulation of lipidated LC3BⅡ in a concentration-dependent manner. Electron microscopy of Marchantin M-treated PC3cells showed the formation of double-or multi-membranes engulfing high electron-density substances, evidenced as autohagosomes or autolysosomes. Marchantin M induced autophagy was also judged by punctate dots of a green fluorescent protein tagged form of LC3B (GFP-LC3B). The cells transfected with GFP-LC3B expression plasmid showed diffuse green fluorescence, while Marchantin M treatment caused significantly punctated green fluorescence in cytosol. The similar results were observed in GFP-LC3ransfected U87cells.3. Mar promoted autophagic flux in HRPC
Data of gene chip and qRT-PCR demonstated that Mar only dramatically induced LC3B expression, and other ATG genes shwoed insignificant difference. Western blotting displayed Mar not only induced LC3B expression but promotes turnover of LC3BⅠ. As increased LC3BⅠ to LC3BⅡ conversion could occur when autophagy was either stimulated or blocked. To further explore if autophagic flux can be induced by Marchantin M, we chose chloroquine (CQ), an agent that prevents the formation of autolysosomes by inhibiting autophagosome-lysosome fusion and LC3BⅡ degradation. The result showed that Marchantin M-stimulated processing of LC3B was enhanced in the presence of CQ, whereas no detectable change was observed in cells exposed to CQ alone, suggesting that Marchantin M has the potential to promote autophagosome formation. Thus, Marchantin M was effective in inducing a prolonged autophagy.
二、Mar induced autophagic cell death in hormone-independent prostate cancer cells
1. Pharmacological blockade of autophagy rescued Mar-mediated cell death
Mar treatment led to the accumulation of LC3BⅡ, whereas this effect was significantly blocked in the presence of3-MA, and accompanied with an increase in cell viability and reduction in cell death. Similarly, Mar-mediated induction of LC3BⅡ was enhanced in combination with E-64D/Pepstatin A, another autophagy inhibitor that prevents lysosomal enzyme activation, and cell proliferation was significantly restored and cell death was markedly decreased in co-treatment.
2. knock-down of LC3B, Atg5and Atg7attenuated Marchantin M-mediated cell death Knockdown of endogenous levels of Atg5by Atg5-targeting siRNA dramatically increased cell viability and blocked cell death after24and48h transfection followed by Mar treatment. Mar-induced LC3BII levels were markedly suppressed when Atg5was down-regulated by siRNA. Similar to the observations of Atg5, reduction of LC3B or Atg7by siRNA significantly alleviated cytotoxic activity of Mar. Taken together, our data clearly emonstrated that Mar activated an autophagic flux and promoted autophagy-dependent cell death.
3. Combined inhibition of caspases and autophagy almost completely rescued Mar-induced cell death
Caspase inhibitors only partially reversed to Mar-induced cell death in hormone-independent prostate cancer cells. Depletion of Atg5expression by siRNA in conjunction with the caspase inhibitor almost completely blocked the cell death induced by mar, which indicated both autophagic cell death and caspase-dependent apoptosis involved in cytotoxicity to hormone-independent prostate cancer cells.
三、PERK/PERK/eIF2α和PI3K/Akt/mTOR involved in Mar-mediated autophagy
1. Mar inhibited PI3K/Akt/mTOR signal by preventing Aktl transcription resulting in autophagy activation.
Western blotting results showed that Mar decreased the phosphorylation level of PDK1, Akt and total Akt protein resulting in reducing phospho-mTOR and phospho-p70S6K. The attenuation of AKT by Mar was further confirmed at mRNA level in PC3cells and the expression of AKT3decreased much lower than that of AKT1/2. Forced expression of AKT-wt was unable to increase the phosphorylation level of GSK3β, a downstream target of AKT, as well as the cell viability exposed to Mar. Unlike to the AKT-wt, PC3cells transfected with AKT-myr showed significant increase in phospho-GSK3β level and cell viability, accompanied with a decrease in the LC3BII. The results showed that Mar inhibited expression and activation of Akt leading to mTOR inactivation and inducing autophagy.
2. Mar-induced ER stress signaling promotes autophagy
Blockade of JNK/c-Jun signal with SP600125hardly affected Mar-induced LC3BII expression and cell death. To explore a link between PERK/eIF2a signaling and autophagic activation in response to proteasome inhibition by Mar, we performed transient transfection with dominant negative PERK (PERK-DN) expression plasmid into cells to impair the function of PERK. The results displayed that Mar-induced eIF2a phosphorylation was blunted by inactivation of PERK. More importantly, co-treatment of cells with Mar and PERK-DN significantly caused the blocking of LC3BII accumulation and partial restoration of viable cells as well as decreased cell death. These results indicate that endoplasmic reticulum stress signal PERK/eIF2a was involved in Mar-mediated autophagy and cell death.
Part IV Conclusions and Innovation
一、Conclusions
1. Mar inhibited proteasome activities in vitro and established PCa cell lines, which indicated Mar was a novel revesible inhibitor of proteasome.
2. Mar disrupted endoplasmic reticulum associated degradation pathway by inhibiting proteasome activity resulting in ER stress, by which promoted cell death in PCa cells.
3. Mar up-regluated ATF3, a stress mediator, in hormone-dependent prostate cancer, which in turn inhibits AR function and promoted apoptosis in LNCaP cells.
4. Mar M indces caspase-dependent and independent cell death in androgen-independent prostate cancer cells by induction of ER stress and autophagy.
二、Innovation and defects
1. We Firstly reported Mar a macrocyclic bisbibenzyls targeted the proteasome activities and showed its inhibitory effect in a reversible manner. Since irreversible inhibitors of proteasome usually cause normal cell apoptosis and death, reversible inhibitors are more valuable in drug discovery. Our data suggests that Mar may represent a promising compound in the future development to treat PCa.
2. We firstly reported Mar prevented AR function by up-regulation of stress mediator ATF3and promoted apoptosis in LNCaP. Since AR in LNCaP cells was mutant, the characteristics of Mar will be beneficial for the treatment of prostate cancer expression constitutive activation of the AR.. However, the effect of Mar on wild-type AR needed to be investigated.
3. Mar triggered autophagic cell death in hormone-independent prostate cancer. Since most chemotherapy agents such as paclitaxel induced autophagic protection in tumor cells, which in turn weakened its anti-tumor effect. Moreover hormone-independent PCa cells showed more potential to anti-apoptosis. However the IC50values of Mar are similar between hormone-dependent and hormone-independent PCa cells and Mar induced autophagic cell death in hormone-independent PCa cells, which conferred Mar a promising anticancer drugs. But animal pharmacodynamics of the compounds needed to be testified.
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