白藜芦醇在原发性脑肿瘤中的代谢特点、活性形式、作用靶点及其与抗癌效果的关系
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摘要
背景与目的髓母细胞瘤(Medulloblastoma,MB)和胶质母细胞瘤(Glioblastoma,GB)分别是好发于儿童和成人的原发性颅内恶性肿瘤。由于它们在颅内呈侵袭性生长,手术难以根除,术后极易复发,故预后甚差。大量的临床资料表明,MB的发病率虽低于白血病,但死亡率却居儿童恶性肿瘤的首位,其对常规放、化疗不敏感,而大剂量的冲击疗法又使患儿难以忍受,即便少数病例经过多方治疗之后得以存活,也经常出现智力发育障碍和在青壮年期间罹患继发性肿瘤以及诸如多囊肾、肺纤维化和发育畸形等多种后遗症。同样,作为星形细胞肿瘤中恶性程度最高的多形性胶质母细胞瘤(Glioblastoma multiforme,GM)即使在手术后接受放射和化学等辅助性治疗,患者一般也只能存活12-15个月,最后死于肿瘤复发。迄今为止,国内外还没有针对这两种原发性脑肿瘤的可靠的辅助性治疗手段。因此,寻求更为有效且无明显毒副作用的MB和GB治疗措施具有明显的学术价值和现实的肿瘤治疗学意义。
以往的研究表明,临床常用的肿瘤细胞分化诱导剂全反式维甲酸(Retinoic acid,RA)虽可诱导髓母细胞瘤细胞系(Med-3)发生分化,但并不能诱导细胞凋亡,且其作用具有明显的可逆性；因此,需要与常规化疗药物如顺铂(DDP)配伍使用才能诱导髓母细胞瘤细胞发生凋亡。我们的近期研究发现,不同髓母细胞瘤细胞系对RA的敏感性有很大不同,其主要原因是细胞内维甲酸结合蛋白-Ⅱ／CRABP-Ⅱ在约60%左右的临床病例中由于DNA甲基化而表达下调甚至沉默,从而影响了RA抗肿瘤信号向胞核内传递。而对于胶质母细胞瘤,单纯RA处理很难抑制其增殖并诱导其死亡,所以需要与其他化学或免疫制剂如INF-γ联用才能达到上述目的。这无疑加大了治疗的毒副作用和对患者的损伤。可见,探索更为安全有效的抗MB和GB药物并阐明其在细胞内的代谢活性形式和抗癌作用的分子机制已成为改善原发性脑恶性肿瘤临床治疗现状的当务之急。
白藜芦醇(Resveratrol, Res)是广泛存在于天然植物如葡萄、草莓、花生中的多酚类次生代谢产物,具有抗心血管疾病、抗突变、抗炎、抗氧化和防治肿瘤等有益的生物药理学活性,并对机体没有毒副作用且能通过血脑屏障。因此,该化合物在防治癌症方面具有独特的药用价值,同时也提示它更适合治疗颅内恶性肿瘤尤其是儿童期脑肿瘤。为此,我门使用白藜芦醇首先对5个不同分化程度的髓母细胞瘤细胞系做了系统的观察。结果显示,白藜芦醇以剂量和时间相关性的方式抑制髓母细胞瘤细胞生长,诱导被处理细胞出现神经元样的表型并死于凋亡；我们还发现,白藜芦醇能上调CYP1A1和下调CYP1B1表达,引起Wnt、Notch和Stat3等信号通路活化状态以及它们下游靶基因表达形式的改变；另外,白藜芦醇能使髓母细胞瘤细胞阻滞在S期并明显下调c-Myc、Survivin和cyclin D1表达。由此可见,白藜芦醇有可能成为临床治疗髓母细胞瘤的理想候选药物。相比之下,胶质母细胞瘤细胞对白藜芦醇的反应却不尽相同,有些敏感如U251细胞系,而更多的则不敏感甚至耐受。故深入探讨导致这种差异发生的内在机制将有助于阐明白藜芦醇治疗胶质母细胞瘤的药用价值,同时为肿瘤个性化治疗的提供理论依据。
尽管白藜芦醇的临床应用价值日趋引起人们的关注,但对白藜芦醇在机体或细胞内的代谢产物活性形式的确定、代谢产物与机体或细胞之间相互作用的机制以及有效代谢产物形成的酶学基础等研究至今仍少见报道,且目前尚无商品化的白藜芦醇代谢物标准品,这些都将成为阻碍白藜芦醇合理用药的瓶颈。目前,在白藜芦醇敏感型细胞内鉴定其有效代谢产物的研究仍十分罕见,而在中枢神经系统肿瘤的研究迄今尚未见报道。可见,开展该领域研究是实现白藜芦醇肿瘤个性化治疗的关键所在,具有明显的转化肿瘤学意义。
鉴于此,本课题将利用髓母细胞瘤细胞系UW228-3和胶质母细胞瘤细胞系LN-18这两个成熟可靠的对白藜芦醇敏感程度迥异的体外实验模型,围绕以下内容展开系统的研究：1)通过高效液相色谱(HPLC)、液质联用技术(LC/MS/MS)及高分辨质谱(HRMS)对白藜芦醇在髓母细胞瘤和胶质母细胞瘤细胞内的代谢产物进行鉴定；2)采用多种分子生物学技术对与代谢产物相关的代谢酶进行分析,以阐明影响药物作用的相关酶的活性状态及表达情况；3)比较白藜芦醇在敏感程度迥异的肿瘤细胞间可能存在的代谢差异,阐明与白藜芦醇抗癌活性密切相关的代谢产物和代谢酶及其亚型和4)探究白藜芦醇处理前后髓母细胞瘤UW228-3和胶质母细胞瘤LN-1 8细胞系中STAT3信号通路活化状态的改变,并进一步分析STAT3上、下游相关基因的表达情况。从而为白藜芦醇抗癌谱之判定和癌症个性化治疗提供科学而可靠的指标。
材料与方法髓母细胞瘤及瘤旁相对正常小脑组织由大连医科大学第一附属医院病理科和中国医科大学盛京医院病理科提供。实验所用人髓母细胞瘤细胞系UW228-3由华盛顿大学(西雅图)神经肿瘤学实验室提供；胶质母细胞瘤细胞系LN-18由瑞士洛桑大学医学院(CHUV)神经外科实验室提供。Wistar大鼠由大连医科大学实验动物中心提供。人髓母细胞瘤UW228-3细胞培养于含有10%胎牛血清的DMEM培养基中,用100gM白藜芦醇处理细胞后,分别收集细胞及培养液；经固相萃取(SPE)纯化后,通过HPLC、LC/MS/MS及高分辨质谱(HRMS)技术,对白藜芦醇在髓母细胞瘤和胶质母细胞瘤细胞中的主要代谢产物及代谢特点进行分析和比较；基于代谢产物的类型,推测出相关代谢酶,利用石蜡组织微阵列技术及免疫组织化学染色(IHC)检测硫酸酯转移酶SULT1A1,1C2和4A 1在人髓母细胞瘤和胶质母细胞瘤中的表达情况,并通过与瘤旁组织及正常鼠脑组织比较(注：由于不可能获取新鲜正常人大脑和小脑组织,故在符合实验动物伦理学原则的情况下,将相应的大鼠脑组织作为正常对照),分析相关代谢酶在正常组织与肿瘤组织中的表达特点；通过流式细胞仪、MTT检测及台盼蓝计数分析不同培养条件下髓母细胞瘤细胞和胶质母细胞瘤细胞对白藜芦醇的敏感程度的变化；采用免疫细胞化学(ICC)、免疫荧光、RT-PCR及Western蛋白印迹分析等方法,检测白藜芦醇处理前后髓母细胞瘤细胞系和胶质母细胞瘤细胞系中相关代谢酶的表达水平及STAT3信号通路活化状态的改变,分析探讨相关靶基因的表达特点。实验数据采用SPSS11.5软件包应用Kruskal-Wallis和Mamm Whitney检验方法进行统计分析。
结果
一、白藜芦醇对髓母细胞瘤UW228-3细胞的作用及其代谢特点
1.白藜芦醇能促进髓母细胞瘤UW228-3细胞分化并诱导其凋亡,抑制其生长
HE及ICC染色结果显示,正常培养的UW228-3细胞呈饱满的梭形,未见突触素表达；经100μM白藜芦醇处理48h后,细胞呈现神经元状的分化表型,细胞中突触素呈阳性表达；流式细胞仪检测的结果显示,UW228-3细胞经100μM白藜芦醇处理后发生G1期阻滞；相应的胎盘蓝染色计数结果表明,UW228-3细胞的染色比例与白藜芦醇作用时间正相关。
2.髓母细胞瘤UW228-3细胞中的白藜芦醇主要代谢产物为单硫酸酯
白藜芦醇在UW228-3细胞中代谢产物经HPLC及LC-MS/MS分析,所得TIC图谱对应的3个色谱峰分别为：M1,反式白藜芦醇,m／z 226.9、m／z 184.9和m／z 1 42.9；M2,顺式白藜芦醇,m/z 226.7、m/z 184.8和m／z 142.9；M3,白藜芦醇单硫酸酯,m/z 307.1、m/z 227.0、m/z 184.9和m／z 143.0。代谢产物进一步通过高分辨质谱(HRMS,阴离子模式)分析得到精确分子质量227.0698(C14H11O3,理论值m／z 227.0708),227.0697(C1 4H11O3,理论值m／z 227.0708)和307.0261(C14H11S06,理论值m／z 307.0276),进一步验证白藜芦醇在UW228-3中代谢产物的类型。其中白藜芦醇不稳定,在自然光下即可产生其异构体顺式白藜芦醇,故M2不是真正意义上的代谢产物。
3.白藜芦醇相关代谢酶SULT 1A1,1C2和4A1在髓母细胞瘤组织中表达水平较瘤旁脑组织及大鼠正常脑组织表达水平低
免疫组织化学染色及Western-blotting结果显示,白藜芦醇相关代谢酶硫酸酯转移酶SULT 1A1,1C2和4A 1在正常脑组织及髓母细胞瘤中均有表达,但SULT 1A1和4A1在肿瘤组织中表达水平与瘤旁组织及大鼠正常小脑组织的表达水平相比有显著性差异(P<0.01); SULT 1C2在髓母细胞瘤患者肿瘤组织表达水平与大鼠正常小脑组织比较有显著性差异(P<0.01),而与瘤旁组织表达水平相比差异不显著(P>0.05)。
4.白藜芦醇虽上调髓母细胞瘤UW228-3细胞硫酸酯转移酶SULT1 A1,1 C2和4A 1表达但总体水平仍低于正常大鼠小脑组织的表达水平
RT-PCR及Western-blotting结果显示,SULT 1C2和4A1在正常培养的UW228-3细胞内表达较低,经1 00μM白藜芦醇处理后其表达水平明显升高,但仍低于正常水平；相对而言,SULT 1A1在正常培养的UW228-3细胞内表达较高,经白藜芦醇处理后表达水平有所升高,但略低于正常组织水平。上述结果在ICC中进一步得到验证。
5.白藜芦醇生物转化产物的抗肿瘤活性低于反式白藜芦醇
利用自然光照射产生的顺式白藜芦醇／白藜芦醇混合物及体外生物转化白藜芦醇单硫酸酯／白藜芦醇混合物(HPLC分析含量均大于50%)分别处理UW228-3细胞,HE及胎盘蓝计数结果显示,UW228-3细胞既无明显的神经元样分化表型,也无明显的细胞凋亡和生长抑现象。
二、白藜芦醇在胶质母细胞瘤LN-18中的代谢特点
1.白藜芦醇处理后的胶质母细胞瘤LN-18细胞无生长抑制和凋亡征象
HE及TUNEL染色结果显示,正常培养的LN-1 8细胞呈椭圆形,100gM白藜芦醇处理48小时后,细胞形态未出现明显的改变,也无明显凋亡；流式细胞仪检测的结果显示,LN-18细胞被白藜芦醇阻滞于G1期；胎盘蓝染色计数结果表明,LN-18细胞的染色比例与白藜芦醇作用时间无明显相关性。
2.白藜芦醇在胶质母细胞瘤LN-18细胞中代谢产物也为白藜芦醇单硫酸酯
HPLC、LC/MS/MS及HRMS的结果显示,白藜芦醇在胶质母细胞瘤LN-18细胞中代谢形式与髓母细胞瘤UW228-3基本相似,即以白藜芦醇单硫酸酯为主。
3.白藜芦醇能够上调胶质母细胞瘤LN-18细胞中硫酸酯转移酶SULT1A1、1C2和4A1的表达水平,上调水平较髓母细胞瘤UW228-3细胞更为明显,但其表达水平仍低于正常大鼠小脑组织表达水平
RT-PCR、Western-blotting及ICC结果显示,胶质母细胞瘤LN-18细胞中除SULT 1A1表达水平较高,SULT 1C2和4A1均呈弱表达；白藜芦醇处理后,SULT 1A1,1C2和4A 1的表达水平均有上调,但SULT4A1的表达依然很弱,而SULT1A1的表达接近正常大鼠小脑组织表达水平。
三、STAT3信号通路在髓母细胞瘤及胶质母细胞瘤中的作用及意义
1.白藜芦醇能够抑制髓母细胞瘤UW228-3细胞中STAT3的转录和活化,并改变STAT3上游和下游基因的表达,促进UW228-3细胞分化和凋亡
RT-PCR及ICC结果显示,STAT3在正常培养的UW228-3细胞的核及浆中均有表达,但经100μM白藜芦醇处理后,STAT3的表达明显下调(P<0.01),在细胞核中几乎检测不到P-STAT3表达；STAT3下游基因如c-Myc、Survivin、Cox-2及cyclin D1经白藜芦醇处理后,表达明显降低,但Bcl-2呈上调趋势；白藜芦醇能够促进STAT3上游基因LIF的表达和分泌；免疫荧光(IF)及ICC结果显示,PIAS3(Protein inhibitor of activated STAT3,活化的STAT3蛋白抑制剂)正常培养的UW228-3细胞中,主要分布于胞浆中,白藜芦醇处理后出现明显的核易位现象,胞浆和胞核均呈阳性表达。
2.不同于敏感的髓母细胞瘤UW228-3细胞,白藜芦醇不抑制胶质母细胞瘤LN-18细胞中STAT3的转录和活化,对STAT3上、下游基因的表达均无显著影响
RT-PCR及Western-blotting结果显示,白藜芦醇处理前后胶质母细胞瘤LN-18细胞中STAT3.p-STAT3及其下游基因c-Myc.Survivin. Cox-2.cyclin D1及Bcl-2的表达无显著性变化(P>0.05):ICC及Western-blotting结果提示,白藜芦醇同样能够促进LN-18细胞中LIF的表达和分泌；ICC及IF结果显示,正常培养条件下,PIAS3在LN-18细胞胞浆中呈弱阳性表达,经白藜芦醇处理后,LN-18细胞胞浆PIAS3表达增强,但胞核依旧呈阴性表达,PIAS3未出现明显核易位现象。
三、STAT3磷酸化抑制剂AG490能够阻断髓母细胞瘤UW228-3及胶质母细胞瘤LN-18中STAT3信号通路,抑制其下游靶基因的转录和活化,60μM AG490能够有效抑制UW228-3和LN-18细胞生长并诱导其凋亡。RT-PCR及ICC结果显示,AG490同时下调c-Myc,Cox-2,cyclin D 1及survivin的表达。
结论
一、白藜芦醇以剂量相关的方式诱导髓母细胞瘤UW228-3细胞的分化和凋亡；但胶质母细胞瘤LN-18细胞对其却不敏感；
二、白藜芦醇在敏感的髓母细胞瘤和耐受的胶质母细胞瘤细胞中的代谢形式基本相同,其代谢产物主要为白藜芦醇单硫酸酯；
三、白藜芦醇作用于UW228-3细胞后10min内即可检测到其代谢产物白藜芦醇单硫酸酯,并于12小时达到峰值,这提示代谢过程先于凋亡现象；
四、硫酸酯转移酶SULT 1A1.1 C2和4A 1主要分布于人和哺乳动物脑中,它在髓母细胞瘤组织中的总体水平要低于瘤旁组织和正常鼠脑组织；
五、白藜芦醇能上调髓母细胞瘤和胶质母细胞瘤细胞中SULT1A1、1 C2和4A 1的表达水平,但表达的总体水平仍低于正常大鼠小脑组织表达水平；
六、白藜芦醇具有化学不稳定性,在自然光下即可转化为其异构体顺式白藜芦醇并导致抗肿瘤活性明显降低；
七、白藜芦醇代谢为白藜芦醇单硫酸酯后,有助于白藜芦醇的胞外排出,从而降低了抗肿瘤效果；
八、白藜芦醇能够有效地抑制髓母细胞瘤的STAT3信号通路并影响STAT3上下游基因的表达,诱导UW228-3细胞凋亡；相反,白藜芦醇对胶质母细胞瘤的STAT3信号通路无抑制作用,LN-18细胞经白藜芦醇处理后无明显凋亡指证。这提示,STAT3对白藜芦醇的反应性因肿瘤细胞的类型而异并与化学敏感性有关；
九、STAT3磷酸化抑制剂AG490能够诱导髓母细胞瘤UW228-3和胶质母细胞瘤LN-18细胞分化和凋亡,同时下调c-Myc,Cox-2,cyclin D 1和survivin的表达水平,从而证明了我们的上述推论；
十、肿瘤细胞SULT的总体水平和STAT3的活化程度与其白藜芦醇敏感性密切相关,可作为白藜芦醇选择性用药和个性化治疗的生物标记物。
Background and objectives:Medulloblastoma (MB) is the commonest primary malignant brain tumor in children, while glioblastoma (GB) is the most common primary brain tumor in adults. The patients with MB and GB have very poor prognosis because of the high recurrent rates due to the difficulty to remove the highly invasive tumor masses radically. A large amount of clinical data showed that MB had the highest mortality rate among childhood malignant tumors although its morbidity rate was lower than leukemia's. It is known that MB is less sensitive to conventional radiotherapy or chemotherapy, and children patient couldn't tolerate stosstherapy with high-dose chemotherapy. Even though some patients survived after multi-treatment, different kinds of sequela such as mental retardation, secondary tumors in his adolescence, ploycystic renal disease, pulmonary fibrosis, developmental malformation could be seen frequently among them. Similarly, patients suffered from glioblastoma multiforme, which is the most malignant tumor in astrocytomas, could only live 12 to 15 months and die of tumor recurrence even though they received adjunctive therapy such as post-operative chemotherapy and radiotherapy. So far, there is still no reliable adjuvant therapeutic means for these two primary brain tumors at home and abroad. Therefore, exploration of more effective and less toxic therapeutic approach for MB and GB is urgently required and is of distinct academic value and clinical significance.
Retinoic acid (RA), as a differentiation inducer, has been commonly used clinically to promote differentiation of certain types of leukemia. However, the data from medulloblastoma Med-3 cells suggest that RA by itself is insufficient to induce apoptosis unless it is used in combination with conventional chemotherapeutic drugs such as cisplatin. We recently found that the sensitivity of MB to RA is largely dependent upon the expression of intra-cellular ratinoic acid binding protein-Ⅱ(CRABP-Ⅱ) and CRABP-Ⅱwas down-regulated or silenced in 60% of MB cases due to DNA Methylation, leading to the defection of RA signal transmission from cytoplasm to the nucleus. Similarly, RA by itself neither inhibits growth nor induces apoptosis of glioblastoma cells; it induces GB cell apoptosis only when combined with immunotherapy agents such as INF-y, which undoubtedly increases the side effects of the treatment and additional injury to the patients. Therefore, it is urgent to explore more effective and safer anti-MB and anti-GB drugs and to analyze their metabolic activity forms and their anti-tumor molecule mechanisms for primary malignant brain tumor treatment, and it would be of clinical significance.
Resveratrol (Res) is a plant polyphenol secondary metabolite existing in grapes, strawberries, peanuts and many other natural foods, which possesses a wide range of biological activities such as cardiovascular protection, antioxidative activity, anti-inflammatory property, and cancer preventive and therapeutic effects. More importantly, this compound has little cytotoxic effect and is able to penetrate blood-brain barrier, suggesting its potential therapeutic values in the management of brain tumors especially intracranial malignancies. We observed systematically five medulloblastoma cell lines in different differentiation degrees, and the results showed that resveratrol could induce differentiation and apoptosis of MB cells in time-and dose-dependent fashions. We also found that resveratrol could up-regulate CYP1A1 but down-regulate CYP1B1 expression, and change the expression of some cancer-associated genes by altering the activities of Wnt, Notch and Stat3 signal pathways. The above evidence suggests that resveratrol may be an ideal drug for MBs in clinical therapy. Compared to MBs, GBs have different responses to resveratrol. Some cell lines such as U251 are sensitive to resveratrol, while others are not sensitive even tolerated to resveratrol. So that, it would be worthwhile to elucidate the internal molecular mechanism leading to different responses to resveratrol, which would help to identify the drug value of resveratrol for GB treatment, and provide a cue for personalized cancer therapy. Although more and more attention has been paid to resveratrol's chemotherapeutic value, it is still unclear whether resveratrol exerts its anticancer effects directly or through its metabolites. So far, no direct evidence has been available concerning the anticancer activity of the parent and conjugated resveratrol because of the difficulty to maintain resveratrol free of metabolism in vivo and to fully transform resveratrol to an identical conjugate in vitro. And this is a neck for resveratrol's rational adminis-tration. As an alternative approach, it would be worthwhile to elucidate the potential therapeutic implications of resveratrol metabolites, So far, the research about identification of resveratol's effective metabolic form in central nervous system's tumour has seldom reported, while studying in this field would be of potential values in exploring tumor-selective and personalized application of resveratrol in cancer prevention and treatment.
In view of this, our work was carried out using a pair of ideal and reliable resveratrol-sensitive MB and insensitive GB tumour cells model.
1) Identification the metabolites in MB and GB cells by HPLC (high performance liquid chromatography), LC/MS/MS (liquid chromatography tandem with mass spectrum) and HRMS (high resolution mass spectrum);
2) Analyse on the metabolic enzyme which responed to resveratrol metabolites by multiple molecular biology skills, to clarify the expression of raleted metabolic enzyme in MB UW229-3 and GB LN-18 cells.
3) Comparing the resveratrol's metabolites and the metabolic enzymes between the MB UW228-3 and GB LN-18 cells, outline the metabolites and the metabolic enzymes which related to resveratrol's anticancer activity;
4) Evaluate the STAT3 signaling and its related gene expression with/without resveratrol's treatment in UW228-3 and LN-18 cells. The findings would be of translational values in exploring individual cancer prevention and treatment of resveratrol.
Materials and methods:Medulloblastoma paraffin embedded speci-mens and their surrounding noncancerous counterparts were provided by the Pathology Department in First Affiliated Hospital of Dalian Medical University and Shengjing Affiliated Hospital of China Medical University. Human MB cell line, UW228-3 was established and kindly provided by the Department of Neurological Surgery, University of Washington at Seattle, USA. GB cell line, LN-18 was provided by Department of Neurological Surgery, University of Lausanne Medical University, Switzerland. Wistar rat was provided by Experimental Animal Centre of Dalian Medical University. Human MB cell line UW228-3 was cultured in Dulbecco's modified Eagles medium (DMEM) containing 10% fetal bovine serum, then the culture media and cells were collected respectively after with/without 100μM resveratrol treatment; After purification by solid phase extraction (SPE), the resveratrol's metabolites in MB and GB cells were separated and identified by HPLC, LC/MS/MS and HRMS; Based on the resveratrol's metabolites, we presumed the related metabolic enzymes sulfotransferases (SULT). The expression of SULT1A1,1C2 and 4A1 were detected in MB and GB tissues by paraffin embedded tissue array and immunohisto-chemistry (IHC), and were compared with the SULTs in normal rat brain tissues for the impossibility to obtain the normal human brain tissues; The chemosensitivity of UW228-3 and LN-18 cells were detected by flow cytometry and trypan blue counts; The SULTs expression in MB and GB cells with/without resveratrol treatment were evaluated by immuno-cytochemistry (ICC), RT-PCR and Western-blotting (WB), then the STAT3 signaling and its upstream/downstream gene expressions were evaluated by RT-PCR, WB, ICC and immunofluorescence (IF). The experimental data were expressed as mean±S.D. and were analyzed with SPSS 11.5 statistic software. Group differences of PCR and WB grey density and viable cell numbers were analyzed by a one-way ANOVA. Mann-Whitney tests were used to analyze the statuses of SULT1A1,1C2 and 4A1 expression in different histological groups. It was considered statistically significant if the p-value is less than 0.05.
Results:
1. Resveratrol-sensitive features and metabolic form in MB UW228-3 cells
1) Resveratrol promoted neuronal-like differentiation and apoptosis of MB UW228-3 cells
HE and ICC results showed that UW228-3 cells were elliptical without synaptophysin expression under normal culture condition; after being treated by 100μM resveratrol for 48 hours, the cells exhibited elongated fibrous phenotype with synaptophysin expression and showed distinct signs of apoptosis. Flow cytometry analyses demonstrated that the G1 and S fractions were 48.50% and 45.97% in normally cultured UW228-3 cells, and changed to 97.62% and 1.26% in the cells treated with 100μM resveratrol for 48h. The percentages of apoptosis in UW228-3 cells were 3.67% before and 31.56% after 48-hour resveratrol treatment. Accordingly, the 0.25% trypan blue evaluation revealed that after 100μM resveratrol treatment for 0, 12,24,36, and 48h, the percentages of unviable cells were 0.01%,11.2%, 30.89%,35.36%, and 42.86% respectively in the UW228-3.
2) Resveratrol monosulfate is the major metabolites in MB UW228-3 cells
Resveratrol's metabolites in UW228-3 cells were separated and identified by HPLC and LC/MS/MS using a combination of full and selected ion scanning techniques. The peaks of M1, M2 and M3 from TIC (total ion chromatogram) represented trans-resveratrol, cis-resveratrol and resveratrol monosulfate, respectively. The negative ion mass spectrum of Ml showed a stable molecular ion at m/z 226.9 which generated m/z 184.9 and m/z 142.9 fragment ion; The spectrum of M2 was identical to M1, and the fragment ions showed m/z at 184.8 and 142.9 generated from m/z 226.7; The [M-H]-ion of M3 showed the deprotonated molecule ions of m/z 307.1 and 227.0, and the m/z 227.0 was fragmented to m/z 184.9 and 143.0, respectively. The identities of the compounds were further confirmed by HRMS that gave the [M-H]- molecular ion exact massas 227.0698 (C14H11O3, caculated m/z 227.0708),227.0697 (C14H11O3, caculated m/z 227.0708) and 307.0261 (C14H11SO6, caculated m/z 307.0276), which corresponded to trans-resveratrol, cis-resveratrol and resveratrol monosulfate, respectively
3) Down-regulated SULTs in medulloblastoma tissues
Immunohistochemical staining and Western blotting showed the three brain-associated SULTs were constitutively expressed in tumour and normal tissues. The expression difference of SULT 1A1 and 4A1 between MB and noncancerous tissues/rat cerebellum was considered statistically signaifi-cant (P<0.01); the distinct difference of SULT 1C2 only existed between MB tissues and rat cerebellum (P<0.01), but the difference between MB and noncancerous tissues was not significant (P>0.05).
4) Resveratrol-enhanced SULTs'expression in MB UW228-3 cells
RT-PCR, Western-blotting and ICC showed in UW228-3 cells, SULT 1C2 was expressed in low and SULT4A1 in extremely low levels, whereas SULT1A1 was expressed in relatively normal level. After resveratrol treatment, the level of SULT1A1 remained almost unchanged, while both SULT4A1 and 1C2 were enhanced, although their levels were still lower than that in the normal cerebella.
5) cis-Resveratrol and resveratrol monosulfate were less bioactive in UW228-3 cells
HE and trypan blue counts showed neither cis-resveratrol enriched s.olution nor resveratrol monosulfate mixture exhibited antimedulloblastoma activity in terms of growth inhibition, neuron-like differentiation and apoptosis induction.
2. Metabolic form in Resveratrol-insensitive glioblastoma LN-18 cells
1) Unlike UW228-3 cells, LN-18 cells showed neither growth arrest nor apoptotic death after resveratrol treatment
HE and TUNEL results showed that LN-18 cells were oval in shape under normal culture condition; 100μM resveratrol treatment for 48 hours didn't cause morphologic change and apoptosis. Flow cytometry analyses demonstrated that the G1 and S fractions were 37.13% and 52.50% in normally cultured LN-18 cells, and changed to 72.85% and 26.72% after 100μM resveratrol treatment for 48h. The 0.25% trypan blue evaluation revealed that the percentages of unviable LN-18 cells were 0.11%,1.20%, 5.63%,8.72% and 10.55% after 100μM resveratrol treatment for 0,12,24, 36, and 48h; accordingly, the percentages of unviable cells in normal culture were 0.09%,0.11%,2.68%,4.66%and 5.69%, respectively.
2) Resveratrol monosulfate is the major metabolites in LN-18 cells
Resveratrol metabolites in LN-18 cells were identified by HPLC, LC/MS/MS and HRMS. The results showed that the major metabolite is resveratrol monosulfate, and its metabolic pattern was similar to UW228-3 cells.
3) Resveratrol up-regulated brain-associated SULTs'expression in GB LN-18 cells
RT-PCR, Western-blotting and ICC showed in LN-18 cells, SULT 1C2 was expressed in low and SULT4A1 in extremely low levels, whereas SULT1A1 was expressed in relatively normal level. After resveratrol treatment, the level of SULT1A1 remained almost unchanged, while both SULT4A1 and 1C2 were enhanced and their overall level was similar than that in the normal cerebella.
3. Effects and significance of STAT3 signaling pathway in MB and GB cells
1) Resveratrol inhibited STAT3 transcription and activation, and altered the expression of STAT3 upstream/downstream genes, which was accompanied with neuron-oriented differentiation
The effects of resveratrol on STAT3 signaling were elucidated by RT-PCR, Western-blotting and ICC. STAT3 was expressed in normally cultured UW228-3 cells, and was down-regulated after resveratrol treatment; STAT3 was distributed in cytoplasm and nuclei of UW228-3 cells under the normal culture condition, which became apparently reduced in the nuclei after resveratrol treatment; Resveratrol altered STAT3 downstream gene expressions, c-Myc, survivin, Cox-2, and cyclin D1 were expressed in normally cultured UW228-3 cells and down-regulated after resveratrol treatment. However, Bcl-2 transcription and production was enhanced by resveratrol incubation, and resveratrol also promoted STAT3 upstream gene LIF expression and secretion; IF and ICC showed that PIAS3 was constitutively expressed and distributed in the cytoplasm, and apparent nuclear translocation of PIAS3 was observed in resveratrol-treated UW228-3 cells.
2) In difference with UW228-3 cells, resveratrol neither inhibited STAT3 transcription and activation nor altered STAT3 upstream/downstream gene expressions in LN-18 cells
The RT-PCR, Western-blotting and ICC results showed that STAT3 was found in both cytoplasmic space and the nuclei of normally cultured LN-18 cells together with c-Myc, survivin, Cox-2 and cyclin D1 expression; this situation remained unchanged after 48h resveratrol treatment. LIF expression was upregulated by resveratrol, while PIAS3 was constitutively expressed and defined in the cytoplasm irrespective to resveratrol treatment.
3) AG490, a selective inhibitor of STAT3 phosphorylation, was used to treat UW228-3 and LN-18 cells. ICC staining demonstrated that STAT3 phosphorylation was inhibited in 60μM AG490-treated cells in terms of the reduction of p-STAT3 nuclear labeling. Meanwhile, the proliferations of UW228-3 and LN-18 cells were apparently suppressed by AG490 and most of the treated cells died of apoptosis after a 48-hour AG490 treatment. RT-PCR and ICC staining revealed that the expression levels of c-Myc, Cox-2, cyclin D1 and survivin were decreased in AG490-treated cells.
Conclusions:
1. Resveratrol could promote MB UW228-3 cells neuronal-like differentiation, and induce cells apoptotic death; However, LN-18 cells showed neither neuronal-like differentiation nor apoptotic death after resveratrol treatment.
2. The resveratrol metabolic pattern in resveratrol-sensitive UW228-3 cells was similar to resveratrol-insensitive LN-18 cells, and its major metabolite was resveratrol monosulfate.
3. The resveratrol monosulfate could be detected in UW228-3 cells as early as 10 min of drug treatment, and its intracellular amount increased to the platform level at 12-h time point, which suggesting that the metabolic machinery pre-existed in the tumor cells and its efficiency was enhanced in response to resveratrol treatment.
4. SULT 1A1,1C2 and 4A1 were preferably expressed in human and rodent brains, and the overall bevels of three brain-associated SULTs in MB tissues were lower than that in human noncancerous tumour-surrounding cerebella, rat normal cerebella as well as LN-18 cells.
5. Resveratrol could up-regulate the three brain-associated SULT 1A1, 1C2 and 4A1 expression in UW228-3 and LN-18 cells, but their overall level in UW228-3 cells was still lower than that in the rat normal cerebella.
6. Resveratrol was chemically unstable and formed an isomer cis-resveratrol in natural light, the cis-form reduced the anti-meullo-blastoma efficacy in terms of less differentiation and apoptosis tendencies in UW228-3 cells.
7. Resveratrol monosulfate, a major metabolite in resveratrol-sensitive UW228-3 and resveratrol-insensitive LN-18 cells, exhibited attenuated cell crisis, which might refleect an active extracelluar excretion due to the increased the solubility of resveratrol.
8. Resveratrol inhibited STAT3 transcription and activation, and altered STAT3 upstream/downstream gene expressions, thus leading UW228-3 cells to neuron-oriented differentiation. But the situation in UW228-3 cells was different because resveratrol neither inhibited STAT3 transcription and activation nor altered STAT3 upstream/downstream gene expressions.
9. AG490 inhibited STAT3 phosphorylation and induced UW228-3 and LN-18 cells to apoptotic death, accompanied with decreased expression levels of c-Myc, Cox-2, cyclin D1 and survivin in both cell lines.
10. Taken together, the metabolic efficiency and the respond manner of STAT3 to resveratrol would be the two parameters for evaluating the chemosensitivities of primanry brain cancer cells to resveratrol and, therefore, would be of translational values in selective application and personalized therapy of resveratrol.
以往的研究表明,临床常用的肿瘤细胞分化诱导剂全反式维甲酸(Retinoic acid,RA)虽可诱导髓母细胞瘤细胞系(Med-3)发生分化,但并不能诱导细胞凋亡,且其作用具有明显的可逆性；因此,需要与常规化疗药物如顺铂(DDP)配伍使用才能诱导髓母细胞瘤细胞发生凋亡。我们的近期研究发现,不同髓母细胞瘤细胞系对RA的敏感性有很大不同,其主要原因是细胞内维甲酸结合蛋白-Ⅱ／CRABP-Ⅱ在约60%左右的临床病例中由于DNA甲基化而表达下调甚至沉默,从而影响了RA抗肿瘤信号向胞核内传递。而对于胶质母细胞瘤,单纯RA处理很难抑制其增殖并诱导其死亡,所以需要与其他化学或免疫制剂如INF-γ联用才能达到上述目的。这无疑加大了治疗的毒副作用和对患者的损伤。可见,探索更为安全有效的抗MB和GB药物并阐明其在细胞内的代谢活性形式和抗癌作用的分子机制已成为改善原发性脑恶性肿瘤临床治疗现状的当务之急。
白藜芦醇(Resveratrol, Res)是广泛存在于天然植物如葡萄、草莓、花生中的多酚类次生代谢产物,具有抗心血管疾病、抗突变、抗炎、抗氧化和防治肿瘤等有益的生物药理学活性,并对机体没有毒副作用且能通过血脑屏障。因此,该化合物在防治癌症方面具有独特的药用价值,同时也提示它更适合治疗颅内恶性肿瘤尤其是儿童期脑肿瘤。为此,我门使用白藜芦醇首先对5个不同分化程度的髓母细胞瘤细胞系做了系统的观察。结果显示,白藜芦醇以剂量和时间相关性的方式抑制髓母细胞瘤细胞生长,诱导被处理细胞出现神经元样的表型并死于凋亡；我们还发现,白藜芦醇能上调CYP1A1和下调CYP1B1表达,引起Wnt、Notch和Stat3等信号通路活化状态以及它们下游靶基因表达形式的改变；另外,白藜芦醇能使髓母细胞瘤细胞阻滞在S期并明显下调c-Myc、Survivin和cyclin D1表达。由此可见,白藜芦醇有可能成为临床治疗髓母细胞瘤的理想候选药物。相比之下,胶质母细胞瘤细胞对白藜芦醇的反应却不尽相同,有些敏感如U251细胞系,而更多的则不敏感甚至耐受。故深入探讨导致这种差异发生的内在机制将有助于阐明白藜芦醇治疗胶质母细胞瘤的药用价值,同时为肿瘤个性化治疗的提供理论依据。
尽管白藜芦醇的临床应用价值日趋引起人们的关注,但对白藜芦醇在机体或细胞内的代谢产物活性形式的确定、代谢产物与机体或细胞之间相互作用的机制以及有效代谢产物形成的酶学基础等研究至今仍少见报道,且目前尚无商品化的白藜芦醇代谢物标准品,这些都将成为阻碍白藜芦醇合理用药的瓶颈。目前,在白藜芦醇敏感型细胞内鉴定其有效代谢产物的研究仍十分罕见,而在中枢神经系统肿瘤的研究迄今尚未见报道。可见,开展该领域研究是实现白藜芦醇肿瘤个性化治疗的关键所在,具有明显的转化肿瘤学意义。
鉴于此,本课题将利用髓母细胞瘤细胞系UW228-3和胶质母细胞瘤细胞系LN-18这两个成熟可靠的对白藜芦醇敏感程度迥异的体外实验模型,围绕以下内容展开系统的研究：1)通过高效液相色谱(HPLC)、液质联用技术(LC/MS/MS)及高分辨质谱(HRMS)对白藜芦醇在髓母细胞瘤和胶质母细胞瘤细胞内的代谢产物进行鉴定；2)采用多种分子生物学技术对与代谢产物相关的代谢酶进行分析,以阐明影响药物作用的相关酶的活性状态及表达情况；3)比较白藜芦醇在敏感程度迥异的肿瘤细胞间可能存在的代谢差异,阐明与白藜芦醇抗癌活性密切相关的代谢产物和代谢酶及其亚型和4)探究白藜芦醇处理前后髓母细胞瘤UW228-3和胶质母细胞瘤LN-1 8细胞系中STAT3信号通路活化状态的改变,并进一步分析STAT3上、下游相关基因的表达情况。从而为白藜芦醇抗癌谱之判定和癌症个性化治疗提供科学而可靠的指标。
材料与方法髓母细胞瘤及瘤旁相对正常小脑组织由大连医科大学第一附属医院病理科和中国医科大学盛京医院病理科提供。实验所用人髓母细胞瘤细胞系UW228-3由华盛顿大学(西雅图)神经肿瘤学实验室提供；胶质母细胞瘤细胞系LN-18由瑞士洛桑大学医学院(CHUV)神经外科实验室提供。Wistar大鼠由大连医科大学实验动物中心提供。人髓母细胞瘤UW228-3细胞培养于含有10%胎牛血清的DMEM培养基中,用100gM白藜芦醇处理细胞后,分别收集细胞及培养液；经固相萃取(SPE)纯化后,通过HPLC、LC/MS/MS及高分辨质谱(HRMS)技术,对白藜芦醇在髓母细胞瘤和胶质母细胞瘤细胞中的主要代谢产物及代谢特点进行分析和比较；基于代谢产物的类型,推测出相关代谢酶,利用石蜡组织微阵列技术及免疫组织化学染色(IHC)检测硫酸酯转移酶SULT1A1,1C2和4A 1在人髓母细胞瘤和胶质母细胞瘤中的表达情况,并通过与瘤旁组织及正常鼠脑组织比较(注：由于不可能获取新鲜正常人大脑和小脑组织,故在符合实验动物伦理学原则的情况下,将相应的大鼠脑组织作为正常对照),分析相关代谢酶在正常组织与肿瘤组织中的表达特点；通过流式细胞仪、MTT检测及台盼蓝计数分析不同培养条件下髓母细胞瘤细胞和胶质母细胞瘤细胞对白藜芦醇的敏感程度的变化；采用免疫细胞化学(ICC)、免疫荧光、RT-PCR及Western蛋白印迹分析等方法,检测白藜芦醇处理前后髓母细胞瘤细胞系和胶质母细胞瘤细胞系中相关代谢酶的表达水平及STAT3信号通路活化状态的改变,分析探讨相关靶基因的表达特点。实验数据采用SPSS11.5软件包应用Kruskal-Wallis和Mamm Whitney检验方法进行统计分析。
结果
一、白藜芦醇对髓母细胞瘤UW228-3细胞的作用及其代谢特点
1.白藜芦醇能促进髓母细胞瘤UW228-3细胞分化并诱导其凋亡,抑制其生长
HE及ICC染色结果显示,正常培养的UW228-3细胞呈饱满的梭形,未见突触素表达；经100μM白藜芦醇处理48h后,细胞呈现神经元状的分化表型,细胞中突触素呈阳性表达；流式细胞仪检测的结果显示,UW228-3细胞经100μM白藜芦醇处理后发生G1期阻滞；相应的胎盘蓝染色计数结果表明,UW228-3细胞的染色比例与白藜芦醇作用时间正相关。
2.髓母细胞瘤UW228-3细胞中的白藜芦醇主要代谢产物为单硫酸酯
白藜芦醇在UW228-3细胞中代谢产物经HPLC及LC-MS/MS分析,所得TIC图谱对应的3个色谱峰分别为：M1,反式白藜芦醇,m／z 226.9、m／z 184.9和m／z 1 42.9；M2,顺式白藜芦醇,m/z 226.7、m/z 184.8和m／z 142.9；M3,白藜芦醇单硫酸酯,m/z 307.1、m/z 227.0、m/z 184.9和m／z 143.0。代谢产物进一步通过高分辨质谱(HRMS,阴离子模式)分析得到精确分子质量227.0698(C14H11O3,理论值m／z 227.0708),227.0697(C1 4H11O3,理论值m／z 227.0708)和307.0261(C14H11S06,理论值m／z 307.0276),进一步验证白藜芦醇在UW228-3中代谢产物的类型。其中白藜芦醇不稳定,在自然光下即可产生其异构体顺式白藜芦醇,故M2不是真正意义上的代谢产物。
3.白藜芦醇相关代谢酶SULT 1A1,1C2和4A1在髓母细胞瘤组织中表达水平较瘤旁脑组织及大鼠正常脑组织表达水平低
免疫组织化学染色及Western-blotting结果显示,白藜芦醇相关代谢酶硫酸酯转移酶SULT 1A1,1C2和4A 1在正常脑组织及髓母细胞瘤中均有表达,但SULT 1A1和4A1在肿瘤组织中表达水平与瘤旁组织及大鼠正常小脑组织的表达水平相比有显著性差异(P<0.01); SULT 1C2在髓母细胞瘤患者肿瘤组织表达水平与大鼠正常小脑组织比较有显著性差异(P<0.01),而与瘤旁组织表达水平相比差异不显著(P>0.05)。
4.白藜芦醇虽上调髓母细胞瘤UW228-3细胞硫酸酯转移酶SULT1 A1,1 C2和4A 1表达但总体水平仍低于正常大鼠小脑组织的表达水平
RT-PCR及Western-blotting结果显示,SULT 1C2和4A1在正常培养的UW228-3细胞内表达较低,经1 00μM白藜芦醇处理后其表达水平明显升高,但仍低于正常水平；相对而言,SULT 1A1在正常培养的UW228-3细胞内表达较高,经白藜芦醇处理后表达水平有所升高,但略低于正常组织水平。上述结果在ICC中进一步得到验证。
5.白藜芦醇生物转化产物的抗肿瘤活性低于反式白藜芦醇
利用自然光照射产生的顺式白藜芦醇／白藜芦醇混合物及体外生物转化白藜芦醇单硫酸酯／白藜芦醇混合物(HPLC分析含量均大于50%)分别处理UW228-3细胞,HE及胎盘蓝计数结果显示,UW228-3细胞既无明显的神经元样分化表型,也无明显的细胞凋亡和生长抑现象。
二、白藜芦醇在胶质母细胞瘤LN-18中的代谢特点
1.白藜芦醇处理后的胶质母细胞瘤LN-18细胞无生长抑制和凋亡征象
HE及TUNEL染色结果显示,正常培养的LN-1 8细胞呈椭圆形,100gM白藜芦醇处理48小时后,细胞形态未出现明显的改变,也无明显凋亡；流式细胞仪检测的结果显示,LN-18细胞被白藜芦醇阻滞于G1期；胎盘蓝染色计数结果表明,LN-18细胞的染色比例与白藜芦醇作用时间无明显相关性。
2.白藜芦醇在胶质母细胞瘤LN-18细胞中代谢产物也为白藜芦醇单硫酸酯
HPLC、LC/MS/MS及HRMS的结果显示,白藜芦醇在胶质母细胞瘤LN-18细胞中代谢形式与髓母细胞瘤UW228-3基本相似,即以白藜芦醇单硫酸酯为主。
3.白藜芦醇能够上调胶质母细胞瘤LN-18细胞中硫酸酯转移酶SULT1A1、1C2和4A1的表达水平,上调水平较髓母细胞瘤UW228-3细胞更为明显,但其表达水平仍低于正常大鼠小脑组织表达水平
RT-PCR、Western-blotting及ICC结果显示,胶质母细胞瘤LN-18细胞中除SULT 1A1表达水平较高,SULT 1C2和4A1均呈弱表达；白藜芦醇处理后,SULT 1A1,1C2和4A 1的表达水平均有上调,但SULT4A1的表达依然很弱,而SULT1A1的表达接近正常大鼠小脑组织表达水平。
三、STAT3信号通路在髓母细胞瘤及胶质母细胞瘤中的作用及意义
1.白藜芦醇能够抑制髓母细胞瘤UW228-3细胞中STAT3的转录和活化,并改变STAT3上游和下游基因的表达,促进UW228-3细胞分化和凋亡
RT-PCR及ICC结果显示,STAT3在正常培养的UW228-3细胞的核及浆中均有表达,但经100μM白藜芦醇处理后,STAT3的表达明显下调(P<0.01),在细胞核中几乎检测不到P-STAT3表达；STAT3下游基因如c-Myc、Survivin、Cox-2及cyclin D1经白藜芦醇处理后,表达明显降低,但Bcl-2呈上调趋势；白藜芦醇能够促进STAT3上游基因LIF的表达和分泌；免疫荧光(IF)及ICC结果显示,PIAS3(Protein inhibitor of activated STAT3,活化的STAT3蛋白抑制剂)正常培养的UW228-3细胞中,主要分布于胞浆中,白藜芦醇处理后出现明显的核易位现象,胞浆和胞核均呈阳性表达。
2.不同于敏感的髓母细胞瘤UW228-3细胞,白藜芦醇不抑制胶质母细胞瘤LN-18细胞中STAT3的转录和活化,对STAT3上、下游基因的表达均无显著影响
RT-PCR及Western-blotting结果显示,白藜芦醇处理前后胶质母细胞瘤LN-18细胞中STAT3.p-STAT3及其下游基因c-Myc.Survivin. Cox-2.cyclin D1及Bcl-2的表达无显著性变化(P>0.05):ICC及Western-blotting结果提示,白藜芦醇同样能够促进LN-18细胞中LIF的表达和分泌；ICC及IF结果显示,正常培养条件下,PIAS3在LN-18细胞胞浆中呈弱阳性表达,经白藜芦醇处理后,LN-18细胞胞浆PIAS3表达增强,但胞核依旧呈阴性表达,PIAS3未出现明显核易位现象。
三、STAT3磷酸化抑制剂AG490能够阻断髓母细胞瘤UW228-3及胶质母细胞瘤LN-18中STAT3信号通路,抑制其下游靶基因的转录和活化,60μM AG490能够有效抑制UW228-3和LN-18细胞生长并诱导其凋亡。RT-PCR及ICC结果显示,AG490同时下调c-Myc,Cox-2,cyclin D 1及survivin的表达。
结论
一、白藜芦醇以剂量相关的方式诱导髓母细胞瘤UW228-3细胞的分化和凋亡；但胶质母细胞瘤LN-18细胞对其却不敏感；
二、白藜芦醇在敏感的髓母细胞瘤和耐受的胶质母细胞瘤细胞中的代谢形式基本相同,其代谢产物主要为白藜芦醇单硫酸酯；
三、白藜芦醇作用于UW228-3细胞后10min内即可检测到其代谢产物白藜芦醇单硫酸酯,并于12小时达到峰值,这提示代谢过程先于凋亡现象；
四、硫酸酯转移酶SULT 1A1.1 C2和4A 1主要分布于人和哺乳动物脑中,它在髓母细胞瘤组织中的总体水平要低于瘤旁组织和正常鼠脑组织；
五、白藜芦醇能上调髓母细胞瘤和胶质母细胞瘤细胞中SULT1A1、1 C2和4A 1的表达水平,但表达的总体水平仍低于正常大鼠小脑组织表达水平；
六、白藜芦醇具有化学不稳定性,在自然光下即可转化为其异构体顺式白藜芦醇并导致抗肿瘤活性明显降低；
七、白藜芦醇代谢为白藜芦醇单硫酸酯后,有助于白藜芦醇的胞外排出,从而降低了抗肿瘤效果；
八、白藜芦醇能够有效地抑制髓母细胞瘤的STAT3信号通路并影响STAT3上下游基因的表达,诱导UW228-3细胞凋亡；相反,白藜芦醇对胶质母细胞瘤的STAT3信号通路无抑制作用,LN-18细胞经白藜芦醇处理后无明显凋亡指证。这提示,STAT3对白藜芦醇的反应性因肿瘤细胞的类型而异并与化学敏感性有关；
九、STAT3磷酸化抑制剂AG490能够诱导髓母细胞瘤UW228-3和胶质母细胞瘤LN-18细胞分化和凋亡,同时下调c-Myc,Cox-2,cyclin D 1和survivin的表达水平,从而证明了我们的上述推论；
十、肿瘤细胞SULT的总体水平和STAT3的活化程度与其白藜芦醇敏感性密切相关,可作为白藜芦醇选择性用药和个性化治疗的生物标记物。
Background and objectives:Medulloblastoma (MB) is the commonest primary malignant brain tumor in children, while glioblastoma (GB) is the most common primary brain tumor in adults. The patients with MB and GB have very poor prognosis because of the high recurrent rates due to the difficulty to remove the highly invasive tumor masses radically. A large amount of clinical data showed that MB had the highest mortality rate among childhood malignant tumors although its morbidity rate was lower than leukemia's. It is known that MB is less sensitive to conventional radiotherapy or chemotherapy, and children patient couldn't tolerate stosstherapy with high-dose chemotherapy. Even though some patients survived after multi-treatment, different kinds of sequela such as mental retardation, secondary tumors in his adolescence, ploycystic renal disease, pulmonary fibrosis, developmental malformation could be seen frequently among them. Similarly, patients suffered from glioblastoma multiforme, which is the most malignant tumor in astrocytomas, could only live 12 to 15 months and die of tumor recurrence even though they received adjunctive therapy such as post-operative chemotherapy and radiotherapy. So far, there is still no reliable adjuvant therapeutic means for these two primary brain tumors at home and abroad. Therefore, exploration of more effective and less toxic therapeutic approach for MB and GB is urgently required and is of distinct academic value and clinical significance.
Retinoic acid (RA), as a differentiation inducer, has been commonly used clinically to promote differentiation of certain types of leukemia. However, the data from medulloblastoma Med-3 cells suggest that RA by itself is insufficient to induce apoptosis unless it is used in combination with conventional chemotherapeutic drugs such as cisplatin. We recently found that the sensitivity of MB to RA is largely dependent upon the expression of intra-cellular ratinoic acid binding protein-Ⅱ(CRABP-Ⅱ) and CRABP-Ⅱwas down-regulated or silenced in 60% of MB cases due to DNA Methylation, leading to the defection of RA signal transmission from cytoplasm to the nucleus. Similarly, RA by itself neither inhibits growth nor induces apoptosis of glioblastoma cells; it induces GB cell apoptosis only when combined with immunotherapy agents such as INF-y, which undoubtedly increases the side effects of the treatment and additional injury to the patients. Therefore, it is urgent to explore more effective and safer anti-MB and anti-GB drugs and to analyze their metabolic activity forms and their anti-tumor molecule mechanisms for primary malignant brain tumor treatment, and it would be of clinical significance.
Resveratrol (Res) is a plant polyphenol secondary metabolite existing in grapes, strawberries, peanuts and many other natural foods, which possesses a wide range of biological activities such as cardiovascular protection, antioxidative activity, anti-inflammatory property, and cancer preventive and therapeutic effects. More importantly, this compound has little cytotoxic effect and is able to penetrate blood-brain barrier, suggesting its potential therapeutic values in the management of brain tumors especially intracranial malignancies. We observed systematically five medulloblastoma cell lines in different differentiation degrees, and the results showed that resveratrol could induce differentiation and apoptosis of MB cells in time-and dose-dependent fashions. We also found that resveratrol could up-regulate CYP1A1 but down-regulate CYP1B1 expression, and change the expression of some cancer-associated genes by altering the activities of Wnt, Notch and Stat3 signal pathways. The above evidence suggests that resveratrol may be an ideal drug for MBs in clinical therapy. Compared to MBs, GBs have different responses to resveratrol. Some cell lines such as U251 are sensitive to resveratrol, while others are not sensitive even tolerated to resveratrol. So that, it would be worthwhile to elucidate the internal molecular mechanism leading to different responses to resveratrol, which would help to identify the drug value of resveratrol for GB treatment, and provide a cue for personalized cancer therapy. Although more and more attention has been paid to resveratrol's chemotherapeutic value, it is still unclear whether resveratrol exerts its anticancer effects directly or through its metabolites. So far, no direct evidence has been available concerning the anticancer activity of the parent and conjugated resveratrol because of the difficulty to maintain resveratrol free of metabolism in vivo and to fully transform resveratrol to an identical conjugate in vitro. And this is a neck for resveratrol's rational adminis-tration. As an alternative approach, it would be worthwhile to elucidate the potential therapeutic implications of resveratrol metabolites, So far, the research about identification of resveratol's effective metabolic form in central nervous system's tumour has seldom reported, while studying in this field would be of potential values in exploring tumor-selective and personalized application of resveratrol in cancer prevention and treatment.
In view of this, our work was carried out using a pair of ideal and reliable resveratrol-sensitive MB and insensitive GB tumour cells model.
1) Identification the metabolites in MB and GB cells by HPLC (high performance liquid chromatography), LC/MS/MS (liquid chromatography tandem with mass spectrum) and HRMS (high resolution mass spectrum);
2) Analyse on the metabolic enzyme which responed to resveratrol metabolites by multiple molecular biology skills, to clarify the expression of raleted metabolic enzyme in MB UW229-3 and GB LN-18 cells.
3) Comparing the resveratrol's metabolites and the metabolic enzymes between the MB UW228-3 and GB LN-18 cells, outline the metabolites and the metabolic enzymes which related to resveratrol's anticancer activity;
4) Evaluate the STAT3 signaling and its related gene expression with/without resveratrol's treatment in UW228-3 and LN-18 cells. The findings would be of translational values in exploring individual cancer prevention and treatment of resveratrol.
Materials and methods:Medulloblastoma paraffin embedded speci-mens and their surrounding noncancerous counterparts were provided by the Pathology Department in First Affiliated Hospital of Dalian Medical University and Shengjing Affiliated Hospital of China Medical University. Human MB cell line, UW228-3 was established and kindly provided by the Department of Neurological Surgery, University of Washington at Seattle, USA. GB cell line, LN-18 was provided by Department of Neurological Surgery, University of Lausanne Medical University, Switzerland. Wistar rat was provided by Experimental Animal Centre of Dalian Medical University. Human MB cell line UW228-3 was cultured in Dulbecco's modified Eagles medium (DMEM) containing 10% fetal bovine serum, then the culture media and cells were collected respectively after with/without 100μM resveratrol treatment; After purification by solid phase extraction (SPE), the resveratrol's metabolites in MB and GB cells were separated and identified by HPLC, LC/MS/MS and HRMS; Based on the resveratrol's metabolites, we presumed the related metabolic enzymes sulfotransferases (SULT). The expression of SULT1A1,1C2 and 4A1 were detected in MB and GB tissues by paraffin embedded tissue array and immunohisto-chemistry (IHC), and were compared with the SULTs in normal rat brain tissues for the impossibility to obtain the normal human brain tissues; The chemosensitivity of UW228-3 and LN-18 cells were detected by flow cytometry and trypan blue counts; The SULTs expression in MB and GB cells with/without resveratrol treatment were evaluated by immuno-cytochemistry (ICC), RT-PCR and Western-blotting (WB), then the STAT3 signaling and its upstream/downstream gene expressions were evaluated by RT-PCR, WB, ICC and immunofluorescence (IF). The experimental data were expressed as mean±S.D. and were analyzed with SPSS 11.5 statistic software. Group differences of PCR and WB grey density and viable cell numbers were analyzed by a one-way ANOVA. Mann-Whitney tests were used to analyze the statuses of SULT1A1,1C2 and 4A1 expression in different histological groups. It was considered statistically significant if the p-value is less than 0.05.
Results:
1. Resveratrol-sensitive features and metabolic form in MB UW228-3 cells
1) Resveratrol promoted neuronal-like differentiation and apoptosis of MB UW228-3 cells
HE and ICC results showed that UW228-3 cells were elliptical without synaptophysin expression under normal culture condition; after being treated by 100μM resveratrol for 48 hours, the cells exhibited elongated fibrous phenotype with synaptophysin expression and showed distinct signs of apoptosis. Flow cytometry analyses demonstrated that the G1 and S fractions were 48.50% and 45.97% in normally cultured UW228-3 cells, and changed to 97.62% and 1.26% in the cells treated with 100μM resveratrol for 48h. The percentages of apoptosis in UW228-3 cells were 3.67% before and 31.56% after 48-hour resveratrol treatment. Accordingly, the 0.25% trypan blue evaluation revealed that after 100μM resveratrol treatment for 0, 12,24,36, and 48h, the percentages of unviable cells were 0.01%,11.2%, 30.89%,35.36%, and 42.86% respectively in the UW228-3.
2) Resveratrol monosulfate is the major metabolites in MB UW228-3 cells
Resveratrol's metabolites in UW228-3 cells were separated and identified by HPLC and LC/MS/MS using a combination of full and selected ion scanning techniques. The peaks of M1, M2 and M3 from TIC (total ion chromatogram) represented trans-resveratrol, cis-resveratrol and resveratrol monosulfate, respectively. The negative ion mass spectrum of Ml showed a stable molecular ion at m/z 226.9 which generated m/z 184.9 and m/z 142.9 fragment ion; The spectrum of M2 was identical to M1, and the fragment ions showed m/z at 184.8 and 142.9 generated from m/z 226.7; The [M-H]-ion of M3 showed the deprotonated molecule ions of m/z 307.1 and 227.0, and the m/z 227.0 was fragmented to m/z 184.9 and 143.0, respectively. The identities of the compounds were further confirmed by HRMS that gave the [M-H]- molecular ion exact massas 227.0698 (C14H11O3, caculated m/z 227.0708),227.0697 (C14H11O3, caculated m/z 227.0708) and 307.0261 (C14H11SO6, caculated m/z 307.0276), which corresponded to trans-resveratrol, cis-resveratrol and resveratrol monosulfate, respectively
3) Down-regulated SULTs in medulloblastoma tissues
Immunohistochemical staining and Western blotting showed the three brain-associated SULTs were constitutively expressed in tumour and normal tissues. The expression difference of SULT 1A1 and 4A1 between MB and noncancerous tissues/rat cerebellum was considered statistically signaifi-cant (P<0.01); the distinct difference of SULT 1C2 only existed between MB tissues and rat cerebellum (P<0.01), but the difference between MB and noncancerous tissues was not significant (P>0.05).
4) Resveratrol-enhanced SULTs'expression in MB UW228-3 cells
RT-PCR, Western-blotting and ICC showed in UW228-3 cells, SULT 1C2 was expressed in low and SULT4A1 in extremely low levels, whereas SULT1A1 was expressed in relatively normal level. After resveratrol treatment, the level of SULT1A1 remained almost unchanged, while both SULT4A1 and 1C2 were enhanced, although their levels were still lower than that in the normal cerebella.
5) cis-Resveratrol and resveratrol monosulfate were less bioactive in UW228-3 cells
HE and trypan blue counts showed neither cis-resveratrol enriched s.olution nor resveratrol monosulfate mixture exhibited antimedulloblastoma activity in terms of growth inhibition, neuron-like differentiation and apoptosis induction.
2. Metabolic form in Resveratrol-insensitive glioblastoma LN-18 cells
1) Unlike UW228-3 cells, LN-18 cells showed neither growth arrest nor apoptotic death after resveratrol treatment
HE and TUNEL results showed that LN-18 cells were oval in shape under normal culture condition; 100μM resveratrol treatment for 48 hours didn't cause morphologic change and apoptosis. Flow cytometry analyses demonstrated that the G1 and S fractions were 37.13% and 52.50% in normally cultured LN-18 cells, and changed to 72.85% and 26.72% after 100μM resveratrol treatment for 48h. The 0.25% trypan blue evaluation revealed that the percentages of unviable LN-18 cells were 0.11%,1.20%, 5.63%,8.72% and 10.55% after 100μM resveratrol treatment for 0,12,24, 36, and 48h; accordingly, the percentages of unviable cells in normal culture were 0.09%,0.11%,2.68%,4.66%and 5.69%, respectively.
2) Resveratrol monosulfate is the major metabolites in LN-18 cells
Resveratrol metabolites in LN-18 cells were identified by HPLC, LC/MS/MS and HRMS. The results showed that the major metabolite is resveratrol monosulfate, and its metabolic pattern was similar to UW228-3 cells.
3) Resveratrol up-regulated brain-associated SULTs'expression in GB LN-18 cells
RT-PCR, Western-blotting and ICC showed in LN-18 cells, SULT 1C2 was expressed in low and SULT4A1 in extremely low levels, whereas SULT1A1 was expressed in relatively normal level. After resveratrol treatment, the level of SULT1A1 remained almost unchanged, while both SULT4A1 and 1C2 were enhanced and their overall level was similar than that in the normal cerebella.
3. Effects and significance of STAT3 signaling pathway in MB and GB cells
1) Resveratrol inhibited STAT3 transcription and activation, and altered the expression of STAT3 upstream/downstream genes, which was accompanied with neuron-oriented differentiation
The effects of resveratrol on STAT3 signaling were elucidated by RT-PCR, Western-blotting and ICC. STAT3 was expressed in normally cultured UW228-3 cells, and was down-regulated after resveratrol treatment; STAT3 was distributed in cytoplasm and nuclei of UW228-3 cells under the normal culture condition, which became apparently reduced in the nuclei after resveratrol treatment; Resveratrol altered STAT3 downstream gene expressions, c-Myc, survivin, Cox-2, and cyclin D1 were expressed in normally cultured UW228-3 cells and down-regulated after resveratrol treatment. However, Bcl-2 transcription and production was enhanced by resveratrol incubation, and resveratrol also promoted STAT3 upstream gene LIF expression and secretion; IF and ICC showed that PIAS3 was constitutively expressed and distributed in the cytoplasm, and apparent nuclear translocation of PIAS3 was observed in resveratrol-treated UW228-3 cells.
2) In difference with UW228-3 cells, resveratrol neither inhibited STAT3 transcription and activation nor altered STAT3 upstream/downstream gene expressions in LN-18 cells
The RT-PCR, Western-blotting and ICC results showed that STAT3 was found in both cytoplasmic space and the nuclei of normally cultured LN-18 cells together with c-Myc, survivin, Cox-2 and cyclin D1 expression; this situation remained unchanged after 48h resveratrol treatment. LIF expression was upregulated by resveratrol, while PIAS3 was constitutively expressed and defined in the cytoplasm irrespective to resveratrol treatment.
3) AG490, a selective inhibitor of STAT3 phosphorylation, was used to treat UW228-3 and LN-18 cells. ICC staining demonstrated that STAT3 phosphorylation was inhibited in 60μM AG490-treated cells in terms of the reduction of p-STAT3 nuclear labeling. Meanwhile, the proliferations of UW228-3 and LN-18 cells were apparently suppressed by AG490 and most of the treated cells died of apoptosis after a 48-hour AG490 treatment. RT-PCR and ICC staining revealed that the expression levels of c-Myc, Cox-2, cyclin D1 and survivin were decreased in AG490-treated cells.
Conclusions:
1. Resveratrol could promote MB UW228-3 cells neuronal-like differentiation, and induce cells apoptotic death; However, LN-18 cells showed neither neuronal-like differentiation nor apoptotic death after resveratrol treatment.
2. The resveratrol metabolic pattern in resveratrol-sensitive UW228-3 cells was similar to resveratrol-insensitive LN-18 cells, and its major metabolite was resveratrol monosulfate.
3. The resveratrol monosulfate could be detected in UW228-3 cells as early as 10 min of drug treatment, and its intracellular amount increased to the platform level at 12-h time point, which suggesting that the metabolic machinery pre-existed in the tumor cells and its efficiency was enhanced in response to resveratrol treatment.
4. SULT 1A1,1C2 and 4A1 were preferably expressed in human and rodent brains, and the overall bevels of three brain-associated SULTs in MB tissues were lower than that in human noncancerous tumour-surrounding cerebella, rat normal cerebella as well as LN-18 cells.
5. Resveratrol could up-regulate the three brain-associated SULT 1A1, 1C2 and 4A1 expression in UW228-3 and LN-18 cells, but their overall level in UW228-3 cells was still lower than that in the rat normal cerebella.
6. Resveratrol was chemically unstable and formed an isomer cis-resveratrol in natural light, the cis-form reduced the anti-meullo-blastoma efficacy in terms of less differentiation and apoptosis tendencies in UW228-3 cells.
7. Resveratrol monosulfate, a major metabolite in resveratrol-sensitive UW228-3 and resveratrol-insensitive LN-18 cells, exhibited attenuated cell crisis, which might refleect an active extracelluar excretion due to the increased the solubility of resveratrol.
8. Resveratrol inhibited STAT3 transcription and activation, and altered STAT3 upstream/downstream gene expressions, thus leading UW228-3 cells to neuron-oriented differentiation. But the situation in UW228-3 cells was different because resveratrol neither inhibited STAT3 transcription and activation nor altered STAT3 upstream/downstream gene expressions.
9. AG490 inhibited STAT3 phosphorylation and induced UW228-3 and LN-18 cells to apoptotic death, accompanied with decreased expression levels of c-Myc, Cox-2, cyclin D1 and survivin in both cell lines.
10. Taken together, the metabolic efficiency and the respond manner of STAT3 to resveratrol would be the two parameters for evaluating the chemosensitivities of primanry brain cancer cells to resveratrol and, therefore, would be of translational values in selective application and personalized therapy of resveratrol.
引文
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21. Gescher AJ, Steward WP. Relationship between mechanisms, bioavailibility, and preclinical chemopreventive efficacy of resveratrol:a conundrum. Cancer Epidemiol Biomarkers Prev.2003;12:953-957.
22. Kirimlioglu V, Ara C, Yilmaz M, Ozgor D, Isik B, Sogutlu G, Kirimlioglu H, Karabulut AB, Yilmaz S, Kayaalp C, Yologlu S. Resveratrol, a red wine constituent polyphenol, protects gastric tissue against the oxidative stress in cholestatic rats. Dig Dis Sci.2006;51:298-302.
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