【作者】 张玉宝；

【导师】 李刚；

【作者基本信息】 山东大学 ， 外科学， 2014， 博士

【摘要】 研究背景和目的胶质母细胞瘤(Glioblastoma Multiforme, GBM, WHO IV级)是一种最常见的原发性恶性脑肿瘤,大约占脑胶质瘤(Glioma)的82%。尽管目前治疗脑胶质瘤主要包括了手术切除、辅以放疗和替莫唑胺化疗等多种综合治疗手段,该类患者预后仍旧很差,从确诊之日起中位生存期仅有15个月。越来越多的研究认为,胶质母细胞瘤细胞自身具有的快速增殖、广泛浸润生长及顽强的侵袭性等生物学行为,以及传统药物难以通过血脑屏障、肿瘤对药物的耐药性等多种因素是导致肿瘤复发、治疗失败的主要原因。因此,迫切需要寻找一种高效低毒且具有较好脂溶性的药物以提高患者的生存率。近年来,植物来源的化合物在肿瘤药物研发中发挥着重要的作用。植物来源尤其是传统中药来源的化合物与化学合成药物相比,具有取材方便、价格低廉、毒副作用小的优势,并且可通过调节机体免疫功能、抑制肿瘤生长、抑制肿瘤血管生成、诱导肿瘤细胞凋亡及分化、调节肿瘤细胞信号传导、抑制转移、抑制端粒酶活性和细胞毒作用等多靶点、多途径实现其抗肿瘤作用,遂受到西方科学界认可,引起国内外众多学者和研究机构的高度关注,成为研发新型抗癌药的重要来源。和厚朴酚(3,5-di-(2-propenyl)-1,1-biphenyl-2,2-diol, honokiol, HNK)是从具有几千年治疗历史的中药材厚朴中分离出来的一种天然生物活性高分子化合物,具有抗炎,抗菌,抗血管生成,抗血栓和抗氧化等多种药理活性。近期研究发现和厚朴酚亦具有潜在的抗肿瘤效应,可明显抑制骨髓瘤、结肠癌、白血病、肝癌、胰腺癌、前列腺癌等多种肿瘤细胞增殖,诱导细胞凋亡、自噬、或类凋亡,且毒副作用小。然而,有关HNK对人胶质母细胞瘤细胞作用的实验研究报道很少,且HNK通过何种信号途径抑制胶质母细胞瘤细胞增殖还不甚清楚。本实验的目的是研究中药单体HNK对人脑胶质母细胞瘤细胞增殖、细胞周期及调亡、迁移和侵袭的影响及其体内的抗肿瘤效应,并以MAPK和STAT3信号通路为切入点探讨其分子机制,为HNK作为临床治疗胶质母细胞瘤新药开发提供实验依据和理论依据。方法和结果一、HNK抗人脑胶质母细胞瘤细胞的体外实验研究1.HNK对人脑胶质母细胞瘤细胞增殖和周期的影响采用CCK-8检测不同浓度HNK处理不同时间后对三种胶质母细胞瘤细胞系U87、U25、T98G细胞增殖的影响,并计算细胞增殖抑制率。结果发现HNK对三种胶质瘤细胞的增殖抑制作用在一定范围内呈现时间-剂量依赖性,并且U87、U251细胞对HNK的作用较为敏感,因此采用这两种细胞系进行后续的实验。采用流式细胞术检测细胞周期分布变化,采用RT-PCR检测细胞周期调控相关蛋白p21、p27及cyclin E的mRNA表达水平。流式结果表明HNK能够引起U87及U251两种细胞系阻滞于G0/G1期。RT-PCR结果显示p21、p27基因的mRNA表达水平升高,而cyclin E的mRNA表达水平降低,阐明了细胞周期阻滞机制。2.HNK诱导U87细胞凋亡及其分子机制。光镜下观察及hoeschst33342染色观察HNK处理前后U87细胞形态学变化。结果发现U87细胞经HNK作用后细胞数目减少,细胞出现皱缩、变圆、脱落,部分细胞出现发泡现象,细胞核则呈现核固缩、染色质边集、核碎裂等典型的凋亡细胞核型变化,表明HNK诱导U87细胞凋亡。为了进一步证实U87细胞发生凋亡,采用Annexin V/PI双染实验对细胞凋亡进行定量分析。结果显示10μg/ml HNK作用于U87细胞12、24、48h后,细胞总凋亡率分别为10.3%,25.1%,26.4%,20μg/ml浓度时,细胞总凋亡率变化不大,但坏死细胞比例增加。采用罗丹明123染色检测线粒体膜电位的改变,采用Western blot检测和凋亡相关的Caspase-3,-8,-9剪切片段、Bcl-2家族bax及Bcl-2、Fas、FasL的蛋白表达水平。结果显示U87细胞经HNK作用后,线粒体膜电位明显损耗, caspase-3、-8、-9剪切片段及Fas/FasL蛋白经HNK作用后表达升高,并且Bax蛋白表达呈现升高趋势,Bcl-2表达呈现降低趋势,Bcl-2/Bax比例明显降低。采用泛caspase抑制剂Z-VAD-fmk预处理U87细胞后,再以HNK处理,Western blot检测caspase-3剪切片段,并以流式检测细胞凋亡水平。结果显示caspase-3活性完全阻断的情况下,泛caspase抑制剂Z-VAD-fmk只是部分减弱了HNK诱导的细胞凋亡。为了进一步阐明HNK诱导U87细胞凋亡的分子机制,采用Western blot检测MAPK信号通路中JNK、p-JNK、p38、p-p38、ERK1/2、p-ERK1/2及STAT3信号通路中STAT3、p-STAT3及下游靶基因survivin的蛋白表达。结果表明HNK作用于U87细胞后p38、ERK1/2、STAT3、JNK及磷酸化JNK蛋白表达无明显变化,而磷酸化p38表达明显升高,磷酸化STAT3及磷酸化ERK1/2呈现表达降低的趋势。3.HNK诱导U251发生类凋亡及分子机制研究光镜下观察及hoeschst3342染色观察HNK处理前后U251细胞形态学变化。结果显示U251细胞经HNK作用后发生明显的胞浆空泡化,未出现典型的凋亡形态学改变。胎盘蓝染色呈现据染,表明细胞的细胞膜完整。细胞经hoeschst33342染色后可见部分细胞的细胞核核形不规则、核浓缩、染色高亮(见图4),但未见核碎裂,染色质也未出现凝集,提示我们U251经HNK处理后可能发生了类凋亡。为了排除HNK处理后U251细胞发生凋亡的可能性,采用Annexin V/PI双染检测细胞早期凋亡,发现U251细胞经HNK作用不同时间点后,细胞总凋亡率无明显变化。为了排除自噬,采用Western blot检测了自噬标志物LC3α/β蛋白表达,结果显示该蛋白表达无明显变化,表明HNK不诱导U251细胞自噬。为了明确HNK对U251细胞的作用,采用透射电镜观察U251细胞超微结构的改变,发现U251细胞膜完整,线粒体及内质网明显肿胀,胞浆内充满大量空泡,具有类凋亡的形态特征。泛Capsase抑制剂不能阻断HNK对U251细胞增殖的抑制作用,并且不能阻断HNK引起的U251细胞空泡化。为了进一步阐明HNK诱导U251细胞类凋亡的分子机制,采用Western blot检测MAPK信号通路中JNK、p-JNK、p38、p-p38、ERK1/2、p-ERK1/2及STAT3信号通路中STAT3、p-STAT3及下游靶基因survivin的蛋白表达。结果表明HNK作用于U87细胞后JNK、p38、ERK1/2、STAT3蛋白表达无明显变化,磷酸化STAT3表达呈现降低趋势,而磷酸化p38、磷酸化ERK与磷酸化JNK在短暂的升高后其表达又开始下降。4.HNK对人脑胶质母细胞瘤细胞迁移和侵袭的影响采用细胞划痕实验检测HNK (5μg/ml、10μg/ml)处理前后细胞迁移能力的变化。用10μl移液器吸头在U251及U87单细胞层上做出一道划痕,以低浓度HNK (5μg/ml、10μg/ml)作用24小时后,在倒置微镜下将划痕宽度的变化进行拍照记录。实验结果显示,HNK抑制了两种细胞的迁移。采用Transwell小室实验检测胶质瘤细胞在HNK的作用下侵袭能力的变化。经过24h的侵袭,将Transwell固小室下表面的已穿膜细胞染色并计数,从而得到胶质瘤细胞相对侵袭力的数据。实验结果显示,HNK能抑制U87和U251细胞的侵袭能力。采用Western blot检测MMP-2和MMP-9蛋白水平变化。发现MMP-2及MMP-9表达均下调。二、HNK抗人脑胶质母细胞瘤细胞的体内实验研究建立荷瘤裸鼠模型,观察HNK对荷瘤裸鼠的抑瘤作用,并采用免疫组化检测survivin蛋白的表达水平。结果表明与对照组比较,HNK组裸鼠体重变化不明显(P>0.05)；与对照组比,HNK组瘤体积显著降低(P<0.05)。免疫组化结果显示HNK组肿瘤细胞survivin蛋白表达明显较对照组降低。结论：1.HNK在体外对人脑恶性胶质母细胞瘤细胞具有明显的增殖抑制作用。2.HNK阻滞细胞周期于G0/G1期,其机制与p21、p27表达上调、cyclinE表达下调相关。3.HNK可诱导胶质母细胞瘤细胞产生多种死亡方式：细胞凋亡、类凋亡、坏死。4.HNK可通过caspase-依赖和caspase-非依赖形式诱导U87细胞凋亡。其中caspase-依赖途径包括激活内源和外源性凋亡途径。5.HNK可通过激活p38通路和抑制ERK1/2、STAT3通路抑制U87细胞增殖、诱导凋亡。6.HNK也可通过诱导类凋亡抑制U251细胞增殖,分子机制可能与MAPK信号通路激活及STAT3信号通路抑制有关。7.HNK可通过抑制MMP-2和MMP-9的表达抑制细胞的迁移和侵袭,发挥其抗胶质母细胞瘤作用。8.HNK在体内亦能抑制细胞增殖,其机制与抑制survivin蛋白表达有关。总之,HNK在体内外均能发挥抗脑胶质母细胞瘤活性,具有潜在的临床开发应用价值。更多还原

【Abstract】 Background and ObjectiveGlioblastoma is the most common and lethal type of primary brain tumor, accounting for82%of cases of malignant glioma. Despite mμltimodal treatment including surgical resection, adjuvant TMZ-based chemotherapy and radio therapy, glioblastoma still remains invariably poor prognosis with only15months of the median overall survival time from diagnosis. It is now commonly recognized that rapid proliferation, the widely infiltrative growth and invariably aggressive biological behavior of glioblastoma cells lead to treatment failure and tumor recurrence. Therefore, the development of novel agents with promising therapeutic resμlts and minimized toxicities for the treatment of glioblastoma is urgently needed.In the past decades natural products have played a critical role in the drug discovery and development. Compared to the synthetic molecμles, natural products from plants, notably from traditional Chinese medicine (TCM), are more easily obtained and more cheap, with less toxicity. In addition, natural products from plants have been reported to exhibit antitumor acitivity by enhancing immunine function, inhibiting proliferation, suppressing angiogenesis, inducing apoptosis and differentiation, regμlating signaling pathways, inhibiting the telomerase activity, as well as their cytotoxicity to cancer cells. Therefore, the potential of natural products from plants has been recognized by the scientific community in the Western world and great efforts have been made to systematically investigate the active component of TCM for cancer therapy.Honokiol (3,5-di-(2-propenyl)-1,1-biphenyl-2,2-diol, HNK) is a natural bioactive molecμlar compound isolated from the Magnolia officinalis, a well-known Chinese traditional medicine Accumμlating studies demonstrated that honokiol exerts multiple pharmacological activities including anti-inflammatory, anti-bacterial, anti-angiogenic, anti-thrombocytic and anti-oxidative activity. Moreover, intense mechanistic and preclinical studies indicate that HNK exhibits potent antitumor activity against a variety of human cancer cell lines, including human multiple myeloma, colon cancer cell line RKO, leukemia cell lines HL-60and Molt4B, hepatocellμlar carcinoma SMMC-7721cells lung, pancreatic cancer cell lines MiaPaCa and PC-3, prostate cancer cells and so on, by inhibiting cell growth and inducing apoptosis or autophagy. Furthermore, HNK exhibited no appreciable toxicity. However, few studies have reported the antineoplastic effects of HNK on glioblastoma cells. Apoptosis induced by HNK and its associated pathway have yet to be discovered. In this study, we aim to investigate the therapeutic potential of HNK against glioblastoma cells in vitro, the biological properties of these tumor cells, including their morphology, proliferation, cell cycle, paraptosis, apoptosis, migration and invasion were studied. Moreover, to elucidate the mechanism of HNK treatment on glioblastoma, the key sinaling proteins in MAPK pathway and STAT3signaling pathway were examined. All the data provided the experimental support for the treatment of glioblastoma with HNK in clinic.Methods and ResultsPart1Effect of HNK on anticancer ability in glioblastomas cell lines.1.1Effect of HNK on proliferation of glioblastomas cell lines and cell cycle distribution.To determine the effect of HNK on the cellular proliferation in glioblastomas, CCK-8assay was performed, according to the protocol provided by the manufacturer. We performed dose-response and time course studies in three established cell lines including U251, U87, and T98G. Inhibition rate were calculated. Our resμlts showed that HNK markedly inhibited growth of all three cell lines in a dose-and time-dependent manner from5to40μg/ml HNK at12-72h. Based on the resμlts obtained in terms of cell growth inhibition, we found that U87cells and U251cells were more sensitive to HNK and then were selected for the following steps of our study.The cellular DNA content distribution of U87and U251cells were examined by flow cytometry and the mRNA expression of P21, p27, and cyclin E was determined by RT-PCR. The results showed that HNK induces a slight G0/G1arrest in U87and U251cells. Moreover, altered expression of cyclin E, p21and p27may account for an arrest in G0/G1phase. 1.2Effect of HNK on apoptosis of U87cells.Cell morphology was observeded under light microscopy after treatment with different concentration of HNK for24h. For nuclear morphology, cells were stained with Hoechst33342and were observed by a Nikon ECLIPSE Ti fluorescence microscope (Nikon, Japan) and photographed. It was noted that after HNK treatment, the number of U87cells decreased and a proportion of the cells shrank, detached from the culture plate and became round. Furthermore, membrane blebbing and formation of apoptotic bodies can be found when cells treated with higher concentration of HNK. Accordingly, Hoechst33342staining results showed that the nuclei of control cells were round and large in size, exhibiting homogeneous blue fluorescence. By contrast, parts of cells treated with10μg/ml HNK for24h exhibited condensed or fragmented nuclei which is the character of cell apoptosis. All of these findings suggested that HNK induced apoptosis of U87cells.To further confirm apoptosis-induced by HNK, Annexin V/PI double staining assay was performed by flow cytometry. A time-dependent study indicated that HNK at10μg/ml induced10.3%,25.1%, and26.4%of total apoptotic U87cells after12,24,48h treatment. While at20μg/mg, the ratio of apoptotic cells has no significant change, accompanied with elevated necrotic cells for8.5%,15.3%,45.4%.Mitochondrial transmembrane potential was monitored by flow cytometry using Rh123. Western blot was performed to detect the activities of effector caspase (caspase-3) and initiator caspase (caspase-8,-9), Bcl-2family proteins (Bcl-2,Bax),as well as Fas/FasL. The results showed that HNK-induced apoptosis is characterized by the activation of caspase-3,-8, and-9, loss of△Ψ, accompanied with elevated expression of Fas and FasL. In addition, HNK increased expression of Bax and decreased expression of Bcl-2, finally resulted in down-regulation of Bcl-2/Bax ratio.To verify whether caspases are necessary in apoptosis induced by HNK in U87cells, we pretreated cells with pan-caspase inhibitor Z-VAD-fmk prior to HNK exposure, then detected the cleaved caspase-3protein and the changes of apoptosis. We found though caspase-3activity was almost totally blocked by the pan-caspase inhibitor Z-VAD-FMK, the inhibitor does not block honokiol-induced apoptosis.We analyzed the activation of intracellμlar pathways related to glioblastomas development MAPK pathways and STAT3. Western blot was performed to detect the protein phosphorylation state of ERK, p38and JNK, STAT3in control cells and in cells treated for1,6,12h with HNK.There were no detectable changes in expression of total ERK, p38, JNK and phosphorylated-JNK protein level. By contrast, phosphorylation of ERK and STAT3were reduced with the time of HNK treatment within12h and obviously declined. Conversely, the treatment caused a significant elevation of the activation of p38..3Effect of HNK on paraptosis of U251cellsCell morphology was observeded under light microscopy after treatment with different concentration of HNK. For nuclear morphology, U251cells were stained with Hoechst33342and were observed by a Nikon ECLIPSE Ti fluorescence microscope (Nikon, Japan) and photographed. It was noted that HNK induced a dose-dependent cytoplasmic vacuolization in U251cells, fitted the criteria of paraptosis or autophagy. The trypan blue dye exclusion assay was perfomed to measure membrane integrity. Hoechst33342staining results showed that although HNK-induced nuclear condensation was observed, but without nuclear fragmentation, lack of apoptotic morphology.To exclude the possibility of apoptosis, Annexin V/PI double staning was performed by flow cytometry. The results exhibited no obvious apoptotic change in U251cells.To exclude the possibility of autophagy, marker of autophagy such as transformation of microtubule-associated protein1light chain3(LC3) Ⅰ to LC3Ⅱ was identified.Western blot results revealed that no significant LC3protein mordification after treatment with HNK.To further confirm the effect of HNK on U251cells, the ultra-structur of U251cells after treament with HNK was observed usinga Transmission Electron.The results revealed that HNK induced extensive cytoplasmic vacuolization in U251cells, swelling of endoplasmic reticulum (ER) and mitochondria.In addition, pretreatment with pan caspase inhibitor can’t block the cytoplasmic vacuolization and the cell growth inhibition induced by HNK.We analyzed the activation of intracellμlar pathways related to glioblastomas development MAPK pathways and STAT3. Western blot was performed to detect the protein phosphorylation state of ERK, p38and JNK, STAT3in control cells and in cells treated for1,6,12h with HNK. there were no detectable changes in expression of total ERK, p38, JNK protein level. By contrast, phosphorylation of STAT3were reduced with the time of HNK treatment within12h and obviously declined. Conversely, the treatment caused a significant elevation of the activation of p38,ERK and JNK.1.4Effect of HNK on migration of and invasion of glioblastoma cells U87and U251.The wound healing assay and Transwell invasion assay were used to assess the effects of HNK on the migration and invasion capacity of U87and U251cells. The sites of the wound line were photographed immediately after scratching and24h later. The width of the wound line was measured, and the migration distances were tocalculated relative to the control group. According to our data, HNK modestly inhibited the motility of U87and U251cells at5ug/ml, and more obviously at lOug/ml. The same results was obtained in Transwell invasion assay.U87and U251cells were serum starved for6h, and treated with various concentration of HNK.24h later, the effect of HNK on MMP-2and MMP-9expression was measured by Western blot. The results showed that the down-regulation of MMP-2and MMP-9were both contributed to migration of and invasion of glioblastoma cells U87and U251.Part2HNK inhibited GBM growth in vivoTo evaluate whether HNK inhibits GBM progression in vivo, human glioblastoma cells U251were injected into the right flank of nude mice. Treatment were started on7days after U251cells injections. HNK and PBS were administered intraperitoneally for14days. Tumor volumes were calculated and the weight of nude rice was detected. Survivin was investigated by ICH. Compared with control group, the weight did not change significantly after treated by HNK(p>0.05); while xenograft tumor growth was significantly slower than the control group after administration; tumor volume was significantly less in HNK treated group than the control group (p<0.05). In addition, The expression of survivin protein in HNK group decreased significantly.ConclusionsIn conclusion, the present study confirmed that HNK inhibited proliferation of glioblastoma cells by causing a slight G0/G1phase cell cycle arrest and inducing apoptosis or paraptosis. Here we demonstrated for the first time that HNK triggered apoptosis of glioblastoma cell line U87through both caspase-independent and caspase-dependent pathways, the latter was initiated with the extrinsic pathway and intrinsic pathway. Moreover, the inhibition of STAT3signaling, ERK1/2as well as activation of p38MAPK signaling pathway may be involed in apoptosis induced by HNK in U87cells.HNK induced paraptosis of U251cells, which were mediated by MAPK signaling and STAT3signaling.HNK also inhibited the cell migration and invasion of glioblastomas cells by down-regulation of MMP-2and MMP-9. In addition, HNK exhibited anticancer ability in glioblastomas cells in vivo. Our findings suggest that HNK treatment could be a promising therapeutic strategy in human glioblastoma.更多还原