【作者】 李欣；

【导师】 邹飞；

【作者基本信息】 南方医科大学 ， 劳动卫生与环境卫生， 2015， 博士

【摘要】 研究背景目的及意义顺铂是常用的肿瘤化疗物质,顺铂与DNA结合形成链内和／或链间链加合物造成遗传物质的损伤,诱发一系列信号蛋白的改变,启动复杂的诱导细胞死亡的机制：如p53活化、p38 MAPKs和／或JNKs活化,NFκB抑制等。在顺铂诱导肿瘤细胞死亡的过程中p53的作用非常重要。p53可以诱导转录依赖和非转录依赖的凋亡。p53转录依赖细胞凋亡是指p53作为转录因子被活化后调节下游凋亡相关基因的表达从而影响凋亡；p53非转录依赖细胞凋亡是p53蛋白本身在产生后可以直接被转运出核,作用于线粒体或线粒体膜上的凋亡相关蛋白(Bcl-2, Bcl-xl)引起细胞凋亡。细胞内p53的水平在静息状态下都维持在较低水平,p53蛋白形成后就与MDM2结合由细胞核输出到细胞质,被MDM2滞留在细胞质中继而泛素化蛋白酶降解。而反过来MDM2的水平受到p53调控,一个负反馈环形成于MDM2与p53之间,保持了MDM2与p53蛋白之间比率的恒定。在受到外界损伤如顺铂等的刺激后,DNA受损引起p53磷酸化,p53的磷酸化破坏了它与MDM2的结合,使p53释放出来,然后磷酸化的p53从细胞质转移进入细胞核,与相应的启动区域结合,发挥其转录依赖和／或非转录依赖的诱导细胞凋亡的生物学功能。黄酮是一种天然多酚类物质,广泛分布于水果、蔬菜等植物中。研究发现黄酮类物质具有抗炎、抗氧化、抗肿瘤等多种生理和药理功效。体内外试验和流行病学研究都证明摄入大量的黄酮类物质会降低许多肿瘤的发病风险。通常认为黄酮类化合物是肿瘤预防或临床多药联合应用方案的理想物质。白杨素(chrysin)是在中草药、蜂蜜及蜂胶中含量丰富的一种黄酮类物质,化学名5,7-二羟黄酮(5,7-dihydroxyflavone)。具有多种生物学功效如抗炎、抗氧化等,虽然分子机制还不明确,但近年已有学者在动物试验和体外研究中观察到白杨素及其衍生物具有一定的肿瘤预防、抑制肿瘤细胞生长和诱导肿瘤细胞凋亡的潜能。我们已有的研究也发现证实了白杨素可以提高多种人类肿瘤细胞对新型抗肿瘤药物肿瘤坏死因子相关凋亡诱导配体(TRAIL)和肿瘤坏死因子TNF-α的敏感性,提高这两种抗肿瘤药物诱导凋亡的能力。相关的研究都提示我们白杨素具有作为抗肿瘤物质的潜能。白杨素作为肿瘤防治药物单独使用,存在着使用剂量高,毒副作用大的缺点,弱化甚至失去了其作为天然产物健康安全的优势和意义。如果白杨素以小剂量甚至食物用量与其它抗肿瘤药物联合应用,则既能保持其安全健康的天然优势,又可充分发掘其抗肿瘤的生物学功效。已有研究观察到抗肿瘤药物联合黄酮类物质染料木素(genistein)、槲皮素(quercetin)、木犀草素(luteolin)等均能有效增强药物对肿瘤细胞的杀伤力。这种将自然界尤其是食物中的天然成分应用于肿瘤辅助治疗的思路,既传承了祖国医学食疗的理念又因天然物质来源丰富、安全健康、应用方便等优势成为发掘新的化疗药物的理想领域。顺铂的耐药性是其临床应用的最大障碍。顺铂的耐药性实质上是顺铂诱导肿瘤细胞凋亡的失败。因此,联合一些能够提高细胞对顺铂诱导凋亡敏感性的小分子物质是克服顺铂耐药性的一种有效的治疗策略。在此,我们研究证明了白杨素与顺铂联合应用能够有效促进肿瘤细胞死亡,并呈剂量效应关系和时间效应关系(Fig.1)。白杨素联合顺铂促进的肿瘤细胞死亡是caspase依赖的细胞凋亡(Fig.2)。这一研究结果在细胞和分子水平为白杨素联合顺铂的应用价值提供了强有力的支持,更为进一步的分子机制探索提供了平台和线索。P53功能失活是肿瘤细胞产生耐药性的一个重要原因。在对顺铂产生耐受的卵巢癌细胞中不能有效启动p53功能是耐药性产生的重要原因。有学者观察到顺铂能够有效促进含p53的肿瘤细胞凋亡而对p53缺陷的肿瘤细胞诱导凋亡能力则明显减弱；研究人员将顺铂和人重组p53联合应用治疗肿瘤以达到克服耐药性提高顺铂的疗效目的,并取得了可喜的成果。本研究中也获得了和报道一致的结果：p53野生型肝肿瘤细胞Hep G2 (p53 WT)和p53缺失型肝肿瘤细胞Hep 3B (p53 deletion)对白杨素联合顺铂处理的反应性有明显的不同,Hep G2细胞出现明显的细胞凋亡而Hep 3B细胞则未观察到明显的细胞死亡(Fig.3A);在单独顺铂没能活化p53也没能够引起细胞死亡的浓度和时间下,白杨素预处理Hep G2细胞后再联合顺铂,就能够有效磷酸化和活化p53 (Fig.3B); p53活化的结果是上调前凋亡蛋白DR5和Bax表达(Fig.4A)。DR5和Bax都是转录因子p53调控的下游分子,DR5活化可以募集Fas相关死亡域蛋白(FADD)和前caspase-8形成死亡复合体(DISC),活化裂解前caspase-8,然后活化的caspase-8进一步裂解活化下游的caspases-3,最终执行外源凋亡；而Bax能穿透线粒体外膜使线粒体胀大,致使线粒体外膜破裂释放细胞色素c而活化caspase-9,激活细胞内源性凋亡。凋亡抑制蛋白Bcl-2阻断凋亡的作用是通过与Bax结合防止细胞色素c释放入细胞质。细胞色素c释放到细胞质(Fig.4B),caspase-8和caspase-9活化(Fig.4C)在本研究均能观察到。所有的下游分子事件都支持p53在白杨素联合顺铂促进肿瘤细胞凋亡的作用中扮演着重要的角色。P53的功能主要通过复杂的翻译后修饰进行调节,p53翻译后修饰包括磷酸化、泛素化、亚硝酸化、乙酰化、甲基化等多种形式,其中各种不同位点的磷酸化是p53翻译后修饰的主要形式。研究认为MAPKs活化是顺铂引起的p53磷酸化的重要的上游分子。MAPKs是一组可被多种信号激活的丝氨酸／苏氨酸激酶,磷酸化丝氨酸、苏氨酸和酪氨酸。MAPKs包括细胞外调节蛋白激酶(extra cellular regulated protein kinases, ERK)、c-jun氨基末端激酶(c-jun N temlinal kinase, JNK)、p38 MAPK和ERK5。ERK活化对于促进顺铂诱导凋亡、减少顺铂耐药性发挥着重要的前凋亡作用。已有研制证明白杨素能够有效的活化三种MAPKs:JNK, p38和ERK1/2。我们的研究数据显示,顺铂未引起细胞凋亡的浓度也没观察到ERK1/2活化,而当白杨素和顺铂联合应用时肿瘤细胞凋亡增加了(Fig.1)同时也会观察到ERK1/2的磷酸化、活化(Fig.5C and 5D)。为了进一步的证明ERK1/2在白杨素联合顺铂促进肿瘤细胞凋亡中对p53活化的作用,随后在顺铂联合应用白杨素之前用ERK1/2抑制剂U0126预处理细胞1 h,观察当ERK1/2的活化被阻断后p53在15位点的丝氨酸的磷酸化的变化情况,如Fig.5E所示p53在15位点的丝氨酸的磷酸化被部分抑制,磷酸化的p53进入细胞核的量也明显减少,免疫荧光的荧光强度减弱(Fig 5F)。所以我们可以说在某种程度上白杨素预处理Hep G2细胞活化的ERK1/2对p53的累积和活化起着积极地促进作用。根据其他学者的研究结果,p53在15位点的丝氨酸确实可以被ERK1/2磷酸化,白杨素对ERK的影响不同报道结果并不一致,可能因细胞种类,不同种属而存在差异。总之,在体外培养肿瘤细胞中白杨素联合顺铂能够有效促进肿瘤细胞凋亡。白杨素可以已通过ERK1/2活化促进的p53磷酸化稳定p53不被降解,发挥p53诱导肿瘤细胞凋亡的肿瘤抑制蛋白作用,提高顺铂对肿瘤细胞的细胞毒性。尽管距离白杨素真正应用到肿瘤临床治疗中还需要对其抗肿瘤的作用及机制做更多更深入的研究,但本研究的发现为白杨素在肿瘤治疗中的应用又提供了一个新的证据。为顺铂联合用药研究探索了一个可行性更好的方案。目标：1、在细胞、分子多水平、多层面探明白杨素联合顺铂对肿瘤细胞凋亡的促进作用。2、体外初步探索白杨素联合顺铂对肿瘤细胞凋亡促进作用的分子机制,分析白杨素对p53蛋白及其相关信号通路的影响,探索1p53蛋白在白杨素联合顺铂协同诱导肿瘤凋亡作用中的调控机制。3、为白杨素联合顺铂的抗肿瘤作用研究的体内研究提供良好的联合用药方案和理论基础,为体内分子机制研究提供线索。4、为黄酮类等天然资源在肿瘤治疗中的应用提供新的思路,为解决顺铂耐药性和化疗的低敏感性问题提供可行性更强的治疗方案。方法：1、敏感细胞株的选择：选择多种组织来源的人类肿瘤细胞系：肝肿瘤细胞(Hep G2、Hep 3B)、结直肠癌细胞(HCT-116)、鼻咽癌细胞(CNE1)等多种细胞,不同剂量白杨素(5、10、20、40 μM)预处理细胞2h后再联合不同剂量的顺铂(2、5、10 μg/mL),倒置显微镜下观察细胞的形态和存活状态,确定单独顺铂或白杨素不出现细胞死亡而联合两种物质则出现明显细胞死亡增加的细胞为对联合作用敏感的肿瘤细胞。2、细胞死亡和凋亡的定量检测分析：倒置显微镜下观察细胞形态和密度,拍照记录细胞死亡的定量资料；流式细胞术检测亚二倍体峰获得亚二倍体峰在整个细胞周期中的百分比,作为细胞死亡的定量指标；荧光核燃料Hoechst 33342或DAPI.染色识别凋亡特异的核固缩确定凋亡细胞,计数200个细胞中凋亡细胞数,获得凋亡细胞定量指标。3、研究白杨素联合顺铂促进肿瘤细胞凋亡的可能机制：以Western blot免疫印记分析磷酸化和总的p53蛋白在不同时间点的变化情况,并以免疫荧光方法分析磷酸化的p53进入细胞核的量进行分析,明确转录因子p53在白杨素联合顺铂促进肿瘤细胞凋亡中的作用。4、证明白杨素联合顺铂促进肿瘤细胞凋亡效应中p53作为转录因子的作用：以Western blot免疫印记分析p53调控的前凋亡蛋白Bax、DR5及p53相关的凋亡抑制蛋白Bcl-2的变化,外源及内源凋亡途径的启动蛋白caspase-8和caspase-9的活化改变情况,及利用线粒体细胞质分离试剂盒获得的细胞质中细胞色素c量的改变,证明p53在白杨素联合顺铂促进肿瘤细胞凋亡效应中转录因子角色。5、白杨素联合顺铂促进肿瘤细胞凋亡效应中影响p53转录活性的上游分子事件：按PureLink RNA Mini Kit (Ambion)试剂说明数提取HepG2细胞总RNA。OneStep RT-PCR分析p53的表达情况,Western blot免疫印记分析磷酸化和总MDM2和ERKl/2的时间变化规律,探索影响p53转录活性的上游分子变化,并使用ERK1/2抑制剂U0126来证明ERK1/2的活化对p53磷酸化的影响。对白杨素联合顺铂促进肿瘤细胞凋亡的分子机制做深入探讨。6、统计分析：数据采用SPSS 13.统计软件分析处理,实验数据用均数±标准差(mean±SD)表示。两组之间样本均数比较以t检验进行统计学分析,包括两独立样本t检验和单样本t检验。多组间均数比较满足方差齐性以单因素方差分析(one-way ANOVA)进行比较,组间两两比较采用LSD’s法：若方差不齐,采用welch检验,组间两两比较采用Dunnett’s T3法进一步进行统计学分析,p<0.05为有统计学意义。结果：1、白杨素和顺铂联合应用能够有效地促进多种人类肿瘤细胞死亡：顺铂处理三种人类肿瘤细胞株：顺铂分别以不同浓度对肝肿瘤细胞(Hep G2) 5μg/mL、结直肠癌细胞(HCT-116) 10μg/mL、鼻咽癌细胞(CNE1) 10μg/mL处理24 h,倒置显微镜下的形态学观察均未发现明显的细胞死亡,说明顺铂在此剂量下对三种肿瘤细胞均为观察到细胞毒性作用。同样的单独白杨素(Hep G2 40μM, HCT-116 40μM, CNE1 20μM)处理三种人类肿瘤细胞株也未观察到细胞死亡。然而三种人类肿瘤细胞在白杨素预处理2h后以顺铂联合处理24 h均可以在倒置显微镜下观察到肿瘤细胞是大量死亡。这也提示白杨素联合顺铂对肿瘤细胞死亡的促进作用具有一定的广谱性。进一步的流式细胞术分析亚二倍体峰,在Hep G2细胞定量分析结果显示：白杨素联合顺铂对肿瘤细胞死亡的促进作用具有剂量效应和时间效应关系。2、白杨素联合顺铂促进的肿瘤细胞死亡是半胱氨酸天冬氨酸酶(caspase)依赖的细胞凋亡：为明确白杨素联合顺铂促进的肿瘤细胞死亡是细胞凋亡,我们首先用细胞核特异性荧光燃料DAPI染色识别凋亡标志性核固缩。白杨素预处理Hep G2细胞后与顺铂联合处理24 h,可以观察到含有核固缩的凋亡细胞量显著增加(F18.050,P=0.007),这为细胞凋亡提供了形态学证据。为进一步证实以上结论Western blot试验检测细胞凋亡的两个标志性蛋白PARP (Poly(ADP-ribose) poly-merase cleavage)和半胱氨酸天冬氨酸酶(Caspase),结果显示PARP和Caspase-3的前蛋白减少、活化裂解片段出现,而且总Caspase的抑制剂Z-VAD-fmk,能够有效的抑制白杨素和顺铂联合作用引起的细胞死亡(F=85.883,P<0.001)及逆转标志性凋亡蛋白(PARP和Caspase-3)的改变。3、p53在白杨素联合顺铂诱导的肿瘤凋亡作用中发挥重要的作用：因为p53在顺铂诱导的肿瘤细胞凋亡中的重要作用,所以我们比较了白杨素联合顺铂分别处理p53野生型肝肿瘤细胞(Hep G2)和p53缺失肝肿瘤细胞(Hep 3B)诱导凋亡的差异：白杨素联合顺铂处理肿瘤细胞24 h后,细胞核特异性荧光燃料Hoechst33342染色,观察到Hep G2细胞凋亡量显著增加(t=-8.110,P=-0.001)说而Hep 3B未见细胞死亡的显著增加(t=-1.512,P=0.205)。随后的p53蛋白分析可观察到白杨素处理Hep G2细胞6h时p53蛋白水平就开始增加,而此时间点单独顺铂处理的Hep G2细胞中并未观察到p53蛋白水平的改变。更为重要的是白杨素联合顺铂处理对p53蛋白水平影响很显著,尤其是在12 h时间点(F=17.822,P=0.003),这提示p53蛋白在白杨素联合顺铂促进肿瘤细胞凋亡中发挥了作用。15位点丝氨酸磷酸化的p53蛋白水平也显著增加(F=105.565,P<0.001)与总p53蛋白水平的变化一致,且p-p53/p53值也显著提高(F=39.068,P<0.001)。这个结果可以在免疫荧光的结果中得到证实,15位点丝氨酸磷酸化的p53蛋白入核显著增加(F=38.164,P<0.001)。4、白杨素通过影响p53下游的分子事件促进顺铂诱导肿瘤细胞凋亡：为进一步说明p53在白杨素联合顺铂促进肿瘤凋亡中的作用,我们对白杨素联合顺铂处理Hep G2细胞后p53下游的分子靶和生物学结果做了分析。Bax和DR5是两个重要的p53调控的前凋亡蛋白,而凋亡抑制蛋白Bcl-2虽不是p53转录调控的下游蛋白,但p53仍是Bcl-2重要的负调节因子。白杨素联合顺铂处理Hep G2细胞可上调前凋亡蛋白Bax和DR5表达、下调凋亡抑制蛋白Bcl-2表达,灰度分析的结果显示,与单独的白杨素或顺铂比较联合两种物质可以显著的提高Bax的相对灰度(F=39.612, P<0.001; P=0.001, P<0.001)和减低Bcl-2的相对灰度(F=50.575, P<0.001; P<0.001, P<0.001),并使Bax/Bcl-2值也显著提高(F=103.826, P=0.003; P=0.016, P=0.013),进而促进细胞色素C释放入细胞质,caspase-8和caspase-9均被活化,最终启动内源和外源凋亡途径,导致肿瘤细胞凋亡。5、白杨素通过活化ERK促进p53在15位点的丝氨酸磷酸化使p53活化入核发挥作用：如果p53蛋白水平的增加如果是由于基因的上调实现的,那么p53在mRNA水平就会发生改变,但白杨素联合顺铂处理Hep G2细胞后RT-PCR并未能检测出p53转录水平的改变。原癌蛋白MDM2是调节p53水平的重要分子,它与p53蛋白结合促进其泛素化蛋白酶降解,但白杨素联合顺铂处理Hep G2细胞后Western blot分析MDM2和磷酸化MDM2均未观察到明显的改变。以磷酸化为主翻译后修饰对p53稳定性和活化发挥重要作用,而DNA损伤类物质正是主要通过蛋白质磷酸化来影响分子信号通路的。之前的结果已观察到白杨素预处理显著的提高p53磷酸化水平。丝裂原活化蛋白激酶(MAPKs),包括SAPK/JNK、p38和ERK1/2,是磷酸化p53的关键的上游激酶,无论是白杨素联合顺铂处理Hep G2细胞的早期阶段还是整个过程ERK1/2都持续活化,而且单独白杨素也能够活ERK1/2,这和p53活化方式是一致的。ERK1/2抑制剂U0126能够部分阻断白杨素联合顺铂引起的p53在15位点丝氨酸的磷酸化和磷酸化的p53转移入细胞核(P=20.939,P<0.001)。以上的结果均说明白杨素联合顺铂处理通过活化ERK1/2而影响了p53的磷酸化,进而促进肿瘤细胞凋亡。结论：1、白杨素联合顺铂可以有效促进人类肿瘤细胞凋亡。2、白杨素预处理引起的p53蛋白活性增高是白杨素联合顺铂促进肿瘤细胞凋亡的内在机制。3、在白杨素和顺铂联合诱导肿瘤细胞凋亡的促进效应中,白杨素预处理活化了ERK1/2进而磷酸化p53促进p53调节的下游Bax和DR5高表达、Bcl-2低表达,最后启动内源和外源凋亡途径,促进肿瘤细胞凋亡增加。更多还原

【Abstract】 Backgroud Aim and SignificanceCis-Diammine-dichloro-platinumⅡ (cisplatin) is a commonly used chemotherapeutical agent in human cancers such as head and neck, testicular, ovarian, cervical, lung, colorectal and relapsed lymphoma. It usually binds to DNA base pairs, and then complex mechanisms are initiated, such as p53, p38 MAPKs and/or JNKs activation, to induce cell death. The significance of p53 in cisplatin-induced cancer cell death has been emphasized. P53 can mediate apoptosis in transcription-dependent and -independent manner. P53-mediated transcription-dependent apoptosis often directly affects expression of downstream genes that regulate sensitivity to apoptosis The p53 protein itself can move out of the nucleus and act upon the mitochondria or proteins in the mitochondria (Bcl-2, Bcl-xl) to induce transcription-independent apoptosis. In normal unstressed cells, p53 is sequestrated in cytoplasm by MDM2 which promotes p53 degradation via the ubiquitin/proteasome pathway. On condition of DNA damage, such as cisplatin, phosphorylation of p53 impairs the ability of MDM2, then phosphorylated p53 translocates to nucleus and exerts its biofunction.Flavonoids are natural polyphenolic phytochemicals that are ubiquitous in plants. They have been reported to possess anti-cancer and chemopreventive properties in numerous epidemiological studies and animal tests. Usually, flavonoids are regarded as ideal candidates for cancer chemoprevention or associated agents in clinical treatment. Chrysin (5,7-dihydroxyflavone), one of flavones, is widely contained in various plants and foods. It possesses many biological activities, including anti-inflammatory and antioxidative properties. Chrysin also has been observed anti-cancer potential in a diverse range of human cells in vitro and test animals in vivo, though the underlying mechanisms have not been clearly illustrated. We have reported previously that chrysin is capable of sensitizing apoptotic cell death induced by tumor necrosis factor or tumor necrosis factor-related apoptosis-inducing ligand in various human cancer cells, suggesting the potential therapeutic value of chrysin in cancer therapy. If chrysin is regarded as a chemotherapy agent on clinical application, high concentration should be used, accompanid with adverse effects and loss of natural properties, such as safty and healthy. If chrysin could be combinated with other anticancer agents at low concentration, we can not only keep the natural advantage of chrysin, but also make full use of the anticancer potential of chrysin。It has been reported that flavonoids, such as genistein, quercetin, luteolin, can promote the cytotoxicity of chemotherapy agents to cancer cells. Natual products is an ideal field to explore assistant agents for anticancer therapy, which fits Chinese traditional medicine.The resistance of tumor cells to cisplatin is one of the major limitations in cisplatin chemotherapy. In essence, cisplatin resistance usually occurs because of a failure to induce apoptosis. Therefore, combined treatment with other sensitizing agents is an effective strategy to overcome cisplatin resistance. Here, we provide experimental evidences that chrysin can promote cisplatin-induced cell death in dose- and time-dependent manner (Fig.1). And cisplatin-induced cell death promoted by chrysin is caspase-dependent apoptosi (Fig.2).Loss of the function of p53 is a contributing factor to the chemotherapeutic resistance in tumors. In cisplatin-resistant ovarian cancer cells, failure to properly regulate p53 can contribute to the resistance. In this study, we found the consist result, no obvious activation of p53 was observed in Hep G2 cells at the concentration and duration of cisplatin alone treatment, but pretreatment with chrysin can phosphorylate and activate p53 (Fig.3). As the result of p53 activation, up-regulation of pro-apoptotic protein DR5 and Bax were found(Fig.4A). Both DR5 and Bax are transcriptional targets for p53 DR5 activation can recruit Fas-associated death domain(FADD) and pro-caspase-8 to form DISC, then DISC-activated autocleavage of pro-caspase-8 to active caspase-8 allows downstream cleavage of caspases-3, leading to the execution of extrinsic apoptosis. Bax can perforate the outer mitochondrial membrane and make mitochondria swell, leading to rupture of the outer mitochondrial membrane and release of cytochrome c to activate caspase-9. Bcl-2 blocks apoptosis by binding to Bax to prevent the release of the pro-apoptotic protein. Cytochrome c release (Fig.4B), caspase-8 and caspase-9 activation (Fig.4C) had been observed. All downstream molecular events supported the critical role of p53 in the encouraging effect of chrysin on cisplatin-induced apoptosis in cancer cells.P53 protein is regulated mainly through complex networks of post-translational modifications. It is believed that MAPKs activation is an important upstream event for p53 phosphorylation in response to ciaplatin. Mitogen-activated protein kinases (MAPKs), including the stress-activated protein kinase/c-Jun-N-terminal kinase (SAPK/JNK), the p38 mitogen-activated protein kinase (MAPK) and the extracellular signal-regulated knase (ERK), are the key upstream kinases to phosporylate p53. ERK activation plays a pro-death role in promoting apoptosis or attenuating cisplatin resistance, and it has been proved that chrysin could activate JNK, p38 and ERK1/2,. Our data indicated that, at the concentration of cisplatin where no much cell apoptosis and no activation of ERK1/2 could be seen, increases of cell apoptosis and phosphorylation of ERK1/2 could be observed (Fig.4C and 4D) when chrysin were applied together. Fourthermore, U0126, inhibitor of ERKl/2, could partly block the p53 phosphorylation at Ser15 and translocation to nuclear (Fig. 4E and 4F). So to some extent, ERK activated by chrysin should contribute to the accumulation and activation of p53 in Hep G2 cells. According to others researches, phophorylation of p53 at Ser15 is really the target of activated ERK1/2. Influence of chrysin on ERK is inconsistent and it appears to be dependent on the cell type.In summary, chrysin can significantly enhance the anticancer effect of cisplatin in vitro. Chrysin is able to stabilize p53 protein via activating ERK1/2 to promote p53 protein phosphorylation. Based on the critical role of p53 in controlling the cell death processes in response to various cancer therapies, we postulate that chrysin may act as a potent chemosensitizer, especially in cancer with wild-type p53. Although further investigations are needed to elucidate the underlying mechanisms of chrysin on enhancing the anticancer effects of chemotherapeutic agents, data from this study provide novel evidence for the potential clinical application for chrysin in cancer therapy.Objective:1. To investigate the effect of chrysin and cisplatin on apoptosis from the cellular and molecular levels.2. To explore the potential molecular mechanism underlying this phenomenon that chrysin reverses the chemoresistance of human cancer cells to cisplatin in vitro. To analyse the influence of chrysin on p53 and its singnaling pathway and research the role of p53 in the promoting effect of chrysin and cisplatin on apoptosis.3. To provide a good schemes and theoretical basis for the combination of chrysin and cisplatin in vivo studies, and provide clues for the study of molecular mechanism in vivo.4. To pove new ways for application of natural products, such as flavonoids, in chemotherapy. To provide more feasible regiments for reversing chrmoresistance and overcoming low sensitivity of cancer cells to chmotherapy agents.Methods:1. Selection of sensitive cancer cells:The human colorectal cancer cell line HCT-116 and the human liver cancer cell lines HepG2 and Hep 3B were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The human nasopharyngeal carcinoma cell line CNE1 was from Sun Yat-Sen University (Guangzhou, China). All cells were pretreated with chrysin (5、10、20、40 μM), then followed by cisplatin(2、5、10 μg/mL) for periods, morphorlogical changes and cell death were observed under the inverted microscope. Then the sensitive cancer cell line was identified.2. Measurement of cell death and apoptosis:Cells in sub-G1 phase were measured to evaluate cell death rates according to the method previously described. Given that cells in sub-G1 phase can be either apoptotic or necrotic, apoptotic cancer cells were further confirmed by staining with either Hoechst 33342 or DAPI. Typically, apoptotic cells exhibit a characteristic chromatin condensation based on Hoechst 33342 or DAPI staining. Briefly, cancer cells were stained with Hoechst 33342 for 20 min or stained with DAPI after paraformaldehyde fixation, then visualized under an inverted fluorescence microscope. The percentage of apoptotic cells were quantified by counting the cells with condensed nuclei among 200 randomly selected cells.3. The potential mechanisem of the combinative effect of chrysin and cisplatin to promote cell apoptosis was explored:The expression levels of total and phorsphorylated p53 were analyed with Western blotting and the translocation of p-p53 to nucleus was detected with IF, the potential role of p53 to the combination o f chrysin and cispltin to promote the apoptosis of Hep G2 cells was identified.4. P53 plays as a transcript factor during the promotive effect of chrysin and cisplatinThe expression levels of Bax and DR5 regulated by p53, Bcl-2 related to p53, were analyed with Western blotting. The extrinsic and intrinsic apoptosis iniater caspase-8 and caspase-9 were detected with Western blotting. Cytosolic proteins were extracted using the Mitochondria Isolation Kit (Thermo Scientific, USA) according to the manufacturer’s instruction, then The level of cytochrome c in the cytosolic protein samples was evaluated using Western blotting analysis. All data supported that p53 plays as a transcript factor during the promotive effect of chrysin and cisplatin.5. The unpstream molecular affairs of p53 were analysed:Total RNA was isolated from HepG2 cells using the PureLink RNA Mini Kit (Ambion). RT-PCR amplifications were performed using OneStep RT-PCR Kit(QIAGEN) in a T100TM Thermal Cycler (Bio-rad) to detect the mRNA levels of p53. The expression levels of total and phorsphorylated MDM2 and ERK1/2 were analyed with Western blotting and the translocation of p-p53 to nucleus was detected with IF. At last the inhibitor of ERK1/2, U0126, was applied to prove the influence of ERK1/2 on the activation of p53 during the combinative effect of chrysin and cisplatin.6. Statistics:All numerical data obtained from at least three independent experiments were presented as mean ± standard deviation (SD). The differences between two groups were anlyzed using the Student’s t-test, including two-sample t-test for independent samples and one sample t-test. The differneces among distinct groups were evaluated using a one-way ANOVA with LSD’s test (SPSS 13.0), when the homogeneity of variances was meet. Groups compared with Welch and inter-group comparision was signidicant using Dunnett’s T3 when the homogeneity of variances was not meet. A P value<0.05 was considered statistically significant.Results:1. The combination of chrysin and cisplatin promoted the death of human cancer cells HepG2, CNE1, and HCT-116A previous study showed that cisplatin-induced cell death was a relatively slow process, normally requiring 2 to 3 days. In the present study, no significant cell death was observed in the three types of cancer cells when they were treated with cisplatin alone (5 μg/mL for HepG2 cells,10 μg/mL for CNE1, and HCT-116 cells) for 24 h, based on morphological examination using an inverted microscope (Fig.1A). Similarly, no obvious cell death was observed when the cancer cells were treated with chrysin alone (40 μM for HepG2 and CNE1 cells, and 20 μM HCT-116 cells). We next tested the effects of the combination of chrysin and cisplatin on cancer cell death. The effect of the combination of chrysin and cisplatin on the death of Hep G2 cells was evaluated by measuring the percentage of sub-G1 cells using flow cytometry. Our results showed that chrysin alone (20 and 40 μM) and the combination of chrysin (20 or 40 μM) and cisplatin (5 μg/mL) significantly increased the death of Hep G2 cells compared with without any treatment (F=206.228, P<0.001) (Fig. 1B). In addition, the rate of dead Hep G2 cells treated with the combination of chrysin (40 μM) and cisplatin (5 μg/mL) was significantly higher than that of Hep G2 cells treated with chrysin (10-40 μM) or cisplatin (5 μg/mL) alone (P< 0.05) (Fig. 1B). Therefore, the combination of chrysin and cisplatin increased the death of Hep G2 cells at a dose-dependent manner. Similarly, the combination of chrysin (40 μM) and cisplatin (5 μg/mL) significantly increased the death of Hep G2 cells at a time-dependent manner (Fig.1C). Significant increase of cell death was observed in Hep G2 cells after 6 h of co-treatment with chrysin (40 μM) and cisplatin (5 μg/mL) (Fig.1C).2. Caspase-dependent apoptosis was involved in increased death of Hep G2 cells induced by the combination of chrysin and cisplatinPrevious studies reported that cisplatin could induce both apoptosis and necrosis. Given that cells at sub-G1 phase can be either apoptotic or necrotic, we then investigated whether the combination of chrysin and cisplatin induced cell death through apoptosis based on DAPI staining. Hep G2 cells were stained with DAPI and examined under a fluorescent microscope. The treatment with the combination of chrysin and cisplatin resulted in evident shrinkage and chromatin condensation of Hep G2 cells (F=18.050, P=0.007, Fig.2A), which are the characteristic signs of apoptosis. To confirm the apoptosis, we evaluated the expression of PARP and caspase-3, two biochemical markers of apoptosis, in Hep G2 cells. We found that the combination of chrysin and cisplatin caused a decrease of pro-proteins and an increase of PARP and caspase-3 cleavages in a dose-dependent manner in Hep G2 cells (Fig.2B). Consistently, pretreatment of Hep G2 cells with Z-VAD-fink, a pan-caspase inhibitor, efficiently blocked PARP and caspase-3 cleavage (Fig.2B) and reversed the cell death (F=85.883, P<0.001, Fig.2C). Taken together, our results suggest that the combination of chrysin and cisplatin promoted the apoptosis of Hep G2 cells, in which caspase was involved.3. p53 was involved in the apoptosis of Hep G2 cells induced by the combination of chrysin and cisplatinGiven that p53 is the key regulator involved in cisplatin-mediated apoptosis of cancer cells, we compared the apoptosis between wild type Hep G2 cells and Hep3B cells with p53 deletion (http://p53.free.fr/Database/Cancer_cell_lines/HCC.html) induced by the combination of chrysin and cisplatin. Significantly more apoptotic cells were observed in Hep G2 (t=-8.110, P=0.001) cells than in Hep3B (t=-1.512, P=0.205) cells after co-treatment with chrysin and cisplatin (Fig.3A). Next, we examined the expression of p53 in Hep G2 cells treated with chrysin, cisplatin, or a combination of both. The protein level of p53 in Hep G2 cells was significantly increased as early as 6 h after chrysin treatment. However, no significant change of p53 expression was found in cisplatin-treated Hep G2 cells, which may be explained by the low concentration of cisplatin and short treatment time employed in the present study (Fig.3B). Interestingly, the combination of chrysin and cisplatin further increased the protein level of p53 in Hep G2 cells, especially after 12 h of co-treatment.The accumulation of phosphorylated p53 (p-p53) was consistent with increased expression of p53 protein (Fig.3B). Densitometric analysis of p53 and p-p53 protein levels showed that the combination of cisplatin and chrysin significantly increased the expression of p53 and p-p53 (Ser15) after 12 h of co-treatment (F=17.822, P=0.003; F=105.565, P<0.001), which led to the increase of the ratio of p-p53 (Serl5) to p53 (F=39.068, P<0.001). In addition, translocation of the phosphorylated p53 (Ser15) protein to nuclei was also observed when Hep G2 cells were treated with chrysin alone (40 μM), or the combination of chrysin (40 μM) and cisplatin (5 μg/mL) for 12h based on immunofluorescence microscopy (F=38.164, P<0.001, Fig.3C). Taken together, these results suggest that p53 was involved in the apoptosis of Hep G2 cells induced by the combination of chrysin and cisplatin.4. The combination of chrysin and cisplatin promoted the apoptosis of Hep G2 cells through p53 pathwaysThe p53 signaling pathways associated with apoptosis are complicated and involve a large number of molecules. Phosphorylation and activation of p53 by upstream kinases regulate p53 downstream genes and the biological consequences. To further elucidate the mechanisms in which p53 is involved in apoptosis promoted by the combination of chrysin and cisplatin, we examined the downstream molecular targets and biological consequences of p53 activation after Hep G2 cells were treated with the combination of chrysin and cisplatin. Bax and DR5 are two main pro-apoptotic genes targeted by p53. DR5 is a member of the tumor necrosis factor receptor superfamily and initiates extrinsic apoptosis by participating in the formation of death-inducing signaling complex (DISC) to activate caspase-8. In the intrinsic apoptosis pathway, Bax is associated with mitochondria and causes cytochrome c release, which activates pro-caspase-9. In contrast, the anti-apoptotic protein Bcl-2 can block intrinsic apoptosis by binding to Bax to form Bcl-2-Bax heterodimers. While p53 is not the direct transcriptional factor for Bcl-2, it is an important negative regulator for Bcl-2. Increased expressions of Bax and DR5 and reduced expression of Bcl-2 were observed in Hep G2 cells treated with the combination of chrysin and cisplatin. Based on densitometric analysis, the protein levels of Bax and Bcl-2 in Hep G2 cells were significantly increased after 18 h of co-treatment of chrysin and cisplatin (F=39.612, P<0.001; F=50.575, P<0.001, Fig.4A) and the ratio if Bax to Bcl-2 increased too (F=103.826, P=0.003). In addition, we observed the release of cytochrome c to cytosol (Fig.4B) and the activation of caspase-9 and caspase-8 (Fig.4C). The expression changes of proteins involved in the p53 pathways suggest that the p53 pathways were involved in the apoptosis of Hep G2 cells induced by the combination of chrysin and cisplatin.5. p53 was activated by ERK through phosphorylation at Ser15While increased expression of p53 protein was observed in Hep G2 cells treated with the combination of chrysin and cisplatin, no significant change of the mRNA level of P53 gene was detected by RT-PCR assay (Fig.5A). Therefore, we speculate that the change of p53 protein is caused by post-translation modification of the protein. We analyzed the oncoprotein MDM2, a p53-binding protein that promotes p53 degradation. Nevertheless, no significant changes of MDM2 and phosphorylated MDM2 were observed at the early stage of chrysin treatment (Fig.5B). Phosphorylation-dependent post-translational modification plays an important role in p53 stabilization and activation, and the antitumor effects of DNA damaging agents are dominantly governed by phosphorylation events. As shown in Fig 3B and C, dramatic p53 phosphorylation at the Ser15 site was detected after chrysin pretreatment. Mitogen-activated protein kinases (MAPKs), including the stress-activated protein kinase/c-Jun-N-terminal kinase (SAPK/JNK), the p38 mitogen-activated protein kinase (MAPK), and the extracellular signal-regulated kinase (ERK), are the key upstream kinases that phosphorylate and regulate p53. Activation of ERK1/2 was observed in Hep G2 cells during the entire process of co-treatment with chrysin and cisplatin (Fig.5C), as well as chrysin treatment alone. This observation was concurrent with p53 activation in Hep G2 cells treated with chrysin alone as well as the combination of chrysin and cisplatin (Fig.3B and C). Additionally, chrysin alone or the combination of chrysin and cisplatin dramatically activated ERKl/2 at early stages (Fig.5D). We showed that U0126, an inhibitor of ERKl/2, partially blocked p53 phosphorylation at Ser15 (Fig.5E) and inhibited the translocation of phosphorylated p53 to nuclei induced by the combination of chrysin and cisplatin (F=20.939, P<0.001, Fig.5F).Conclusion:1. Combination of chrysin and cisplatin promotes the apoptosis of human cancer cells.2. Enhancement of p53 by pretreatment with chrysin could be a potential molecular mechanism underlying this phenomenon.3. P53 accumulation through activating ERK1/2 by chrysin can promote Bax, DR5 expression and suppress Bcl-2 expression which initiates extrinsic and intrinsic apoptosis in the promoting effect of chrysin and cisplatin.更多还原