【作者】 孙素霞；

【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学， 2011， 博士

【摘要】 背景结肠癌是最常见的恶性肿瘤之一,国际流行病学资料显示,结肠癌的发病率在恶性肿瘤发病率中居第3位,死亡率中居第4位,2008年有1,233,700新发病例和608,700死亡病例,具有发病率高、死亡率高、治愈率低等特点。尽管近年来射靶治疗药物(如西妥昔单抗和贝伐单抗)的出现和其他治疗方法的改进,但结肠癌的预后仍不能令人满意。因此,探寻结肠癌的预防和治疗药物就显得尤为重要。短链脂肪酸主要包括乙酸、丙酸和丁酸,是肠道微生物发酵膳食纤维产生的主要代谢产物,可抑制结肠癌、乳腺癌和淋巴瘤等多种肿瘤细胞的增殖、并促进其凋亡和分化,其中丁酸的作用最为显著,但其作用机制尚不清楚。有研究显示,丁酸钠(sodium butyrate, NaB)是一种组蛋白去乙酰化酶抑制剂(histone deacetylase inhibit, HDACi),可通过抑制组蛋白去乙酰化酶活性,发挥其各种功能。体内外试验表明,多种HDACi具有低毒强抗癌作用以及肿瘤细胞高度选择性等特点。为此,HDACi是近年来抗肿瘤药物研究领域中的热点之一。目前已有多种HDACi,如辛二酰苯胺异羟肟酸(Suberoylanilide hydroxandc acid, SAHA)、丙戊酸(valproic acid, VPA)、曲古抑菌素A (Trichostatin A, TSA)等已进入恶性肿瘤临床Ⅰ期和Ⅱ期试验阶段。2006年,美国FDA批准第一个HDACi药物vorinostat (N-羟基-N’-苯基辛二酰胺)作为抗肿瘤药物,主要用于治疗顽固复发性、耐药性T细胞淋巴癌,并由美国Merck公司生产上市。NaB作为一种典型的HDACi,可通过抑制组蛋白去乙酰化酶活性,引起组蛋白的高度乙酰化,从而上调多种促凋亡蛋白(Bid, Bak, Fas, FasL, TRAIL, caspase)和/或下调多种抗凋亡蛋白(Bcl-2, Bcl-XL, XIAP, survivin, cFLIP)的表达,最后通过线粒体和/或死亡受体途径诱导多种肿瘤细胞凋亡。细胞内钙浓度的升高或下降可引起一系列细胞生物学反应,如神经递质分泌、骨骼肌收缩等快速反应,以及细胞分化、有丝分裂、细胞凋亡等慢速反应。研究显示,无论是在早期还是晚期细胞内钙浓度的升高都有促细胞凋亡作用。钙池调控钙内流(store-operate calcium entry, SOCE)最早被命名为库容性钙内流(capacitative calcium entry, CCE),当时认为内质网钙库中钙离子释放可促发细胞外钙离子通过质膜上的钙通道内流。尽管CCE概念早在1986年就已经发现,但是构成SOCE的关键分子及其机制直到近几年才逐渐被发现。一个是基质相互作用分子(stromal interaction molecule1, STIM1), STIM1被认为是钙离子浓度感受器,主要定位在内质网膜上,仅少量定位于细胞膜；另一个是通道蛋白-Orail, Orail主要定位于细胞膜。内质网钙库中钙离子清空后可引起内质网膜上散在分布的STIM1发生寡聚化并发生移位,靠近细胞膜,进而引起细胞膜上的Orail通道由散在分布形成离散的斑点状分布,通道功能被激活,细胞外钙离子内流。研究表明,通过SOCE的细胞外钙内流参与多种重要的生理功能,如细胞分化、细胞凋亡和细胞增殖等。但SOCE是否在NaB诱导结肠癌细胞凋亡过程中发挥作用,还未有研究报道。因此,本研究主要探讨SOCE是否参与NaB诱导结肠癌细胞凋亡过程,为开发NaB作为肿瘤治疗药物提供理论依据。方法将结肠癌细胞株HCT-116置于37℃培养箱内培养(5%C02、95%空气),分别用不同浓度的NaB处理HCT-116不同时间后,流式细胞仪检测细胞凋亡率,免疫印迹法检测聚腺苷酸二磷酸核糖转移酶(poly ADP-ribose polymerase,PARP,细胞凋亡标志性分子)的切割片段。观察细胞内外钙螯合剂(BAPTA-AM和EGTA)、SOCE非特异性阻断剂(2-APB和SKF96365)以及下调STIM1的表达等预处理对NaB促结肠癌细胞凋亡率及PARP切割片段的影响。用免疫印迹法和免疫荧光法检测NaB对结肠癌细胞STIM1和Orail活性的影响。结果1.NaB可诱导结肠癌细胞凋亡用hoechst33342染色方法检测NaB处理结肠癌细胞HCT-116后细胞核形态变化。结果显示10mM NaB作用结肠癌细胞HCT-11624小时后,HCT-116细胞核出现明显的核浓缩、染色质片段等细胞凋亡的特征性表现,并形成典型的凋亡小体。用不同浓度的NaB处理结肠癌细胞HCT-11624小时后,流式细胞仪检测细胞凋亡率和免疫印迹法检测PARP,结果显示,1.25mM NaB处理24小时后细胞凋亡率和PARP的切割片段与对照组相比明显升高并具有统计学意义(P<0.05)。用10mM NaB作用结肠癌细胞HCT-116不同时间后,流式细胞仪检测细胞凋亡率和免疫印迹法检测PARP,结果显示,24小时后细胞凋亡率和切割PARP明显升高,与对照组比较之间差异具有统计学意义(P<0.01)。以上结果表明,NaB可诱导结肠癌细胞HCT-116凋亡,并具有剂量和时间反应关系。2.螯合细胞内外钙离子对NaB诱导结肠癌细胞凋亡率的影响为探讨钙离子在NaB促结肠癌细胞凋亡过程中的作用,本研究中利用细胞外、内钙螯合剂—EGTA和BAPTA-AM分别预孵HCT-116细胞1小时后,再用10mM NaB处理24小时,流式细胞仪检测细胞凋亡率,免疫印迹法检测PARP。结果显示,0.5mM EGTA和10μM BAPTA-AM预孵组与仅NaB作用组之间细胞凋亡率和PARP切割片段的差异具有统计学意义(P<0.05)。实验结果表明,EGTA和BAPTA-AM预孵可显著降低NaB促结肠癌细胞凋亡率,提示,钙离子在NaB促结肠癌细胞凋亡过程中发挥一定作用。3.阻断SOCE对NaB诱导结肠癌细胞凋亡率的影响为进一步验证钙离子参与NaB诱导结肠癌细胞凋亡过程是否是由SOCE介导,研究中采用SOCE的非特异性抑制剂,即2-APB和SKF96365分别预处理HCT-1161小时后再加入10mM NaB作用24小时,检测75μM2-APB和5μMSKF96365预处理对NaB诱导结肠癌细胞凋亡率的影响。流式细胞仪检测细胞凋亡率,免疫印迹检测PARP切割情况。结果显示,2-APB和SKF96365预处理与仅NaB处理组之间细胞凋亡率和切割片段PARP的差异具有统计学意义(P<0.05)。实验结果表明,SOCE非特异性抑制剂预处理可显著降低NaB诱导结肠癌细胞的凋亡率。为进一步证实SOCE通道在NaB诱导结肠癌细胞凋亡中的作用,我们采用RNA干扰技术,下调SOCE关键分子-STIM1的表达,real-time PCR和免疫印迹法验证干扰效率。结果显示,实验中所设计的两对序列干扰效果均较好,STIM1在mRNA和蛋白水平的表达量均下调至30%左右。STIM1表达下调后再加入10mM NaB处理结肠癌细胞,观察STIM1表达下调对NaB诱导结肠癌细胞凋亡率的影响。结果显示,下调STIM1蛋白可显著降低NaB促结肠癌细胞HCT-116的凋亡率,并减少NaB对PARP的切割效率,STIM1表达下调后再加NaB组与NaB之间细胞凋亡率和切害PARP相对灰度值的差异具有统计学意义(P<0.05)。以上结果表明,STIM1在NaB诱导结肠癌细胞凋亡过程中发挥了一定的作用,从而进一步证实SOCE参与NaB诱导结肠癌细胞的凋亡过程。4.NaB通过调节STIM1的移位并与Orail之间通过共定位作用调节凋亡过程我们用免疫印迹法检测NaB作用结肠癌细胞HCT-116不同时间、以及不同浓度后STIM1表达量的变化,进一步验证NaB对STIM1蛋白表达量的影响。结果显示,不同时间和不同浓度NaB处理后STIM1在蛋白水平的表达量与对照组比较差异不具有统计学意义(P>0.05)。STIM1在蛋白水平的表达量不随NaB作用时间和作用浓度的增加而变化,表明NaB作用结肠癌细胞HCT-116不影响STIM1蛋白的表达量。上述实验结果显示STIM1在NaB诱导结肠癌细胞凋亡的过程中具有一定的作用,但其作用与表达量的多少无关。那么,是否STIM1在NaB诱导结肠癌细胞凋亡中的作用与其位置的移动有关?为此,我们采用免疫荧光技术利用激光共聚焦显微镜观察STIM1和Orai1在NaB作用后位置的变化。结果显示,10mMNaB处理后STIM1位置发生了明显的改变,由全细胞内均匀分布逐渐移向细胞膜,且与Orai1蛋白在细胞膜附近有明显的共定位现象。进一步说明,NaB可通过改变STIM1的定位,与Orai1共定位现象而诱导结肠癌细胞凋亡。结论本研究的实验结果显示,NaB可诱导SOCE的关键分子STIM1的移位并与通道蛋白Orai1发生共定位,从而诱导细胞外钙离子内流,从而促进结肠癌细胞的凋亡,说明SOCE介导的细胞外钙内流在NaB诱导结肠癌细胞凋亡过程中发挥一定作用更多还原

【Abstract】 BackgroundColon cancer is the third most common cancer and the fourth leading cause of cancer-related death in the world, with an estimated incidence of1,233,700new cases and a mortality of608,700deaths annually based on the statistics for the year2008. Despite the recent advent of targeted therapy (e.g. cetuximab and bevacizumab) and the improvement of other treatment modalities, the prognosis for patients with metastatic colon cancer remains limited. This reality highlights a need to develop new chemoprophylactic agents for prevention of colon cancer at the early stage.Short-chain fatty acids have been shown to inhibit progression of colorectal cancer. They are naturally produced via the process of anaerobic microbial fermentation of dietary fibers within the colon. Among them, butyric acid has received the greatest attention due to its potent and pleiotropic anticancer effects. Sodium butyrate inhibits cell cycle progression, induces apoptosis and promotes differentiation in various types of cancer cells, including colorectal cancer, lymphoma cancer and breast cancer. However, the mechanism of the action remains elusive. It was suggested that the anticancer effect of sodium butyrate is mediated, at least in part, through inhibition of histone deacetylase. Ca2+signaling pathway plays an important role in many cellular activities, including neurosecretion, skeletal muscle contraction, cell growth and differentiation. Accumulating evidence supports the notion that increased cytoplasmic Ca2+could cause cell apoptosis, at both early and late stages of apoptotic processes. Store-operated Ca2+entry (SOCE) refers to the phenomenon that depletion of intracellular Ca2+stores activates Ca2+channels in the plasma membrane as a compensatory mechanism to refill the internal Ca2+store, stromal interaction molecule1(STIM1) and Orail are two proteins essential for SOCE. STIM1has a single transmembrane domain and an EF-hand Ca2+binding domain as a sensor of the ER luminal Ca2+. Through redistribution, STIM1binds to and transmits signal to Orail, the pore forming subunit of the store-operated Ca2+(SOC) channel. This interaction results in extracellular Ca2+influx. The SOC channel-mediated Ca2+release from the ER and extracellular Ca2+influx could in turn cause apoptosis. Therefore, since SOCE is one of the major mechanisms for Ca2+entry in non-excitable cells, we hypothesized that SOCE is involved in apoptosis induced by sodium butyrate, however, has not yet been investigated. Here, we report that both intracellular and extracellular Ca2+play a key role in sodium butyrate-induced apoptosis in the colon cancer cell line, HCT-116. Moreover, the influx of extracellular Ca2+is mediated through SOCE.MethodsCell culture, Conventional and Real-Time Reverse Transcription-Polymerase Chain Reaction, Western Blots, Annexin V/PI assay for apoptosis, Morphological assessment of apoptosis, RNA Interference, Immunofluorescence Imaging.Results 1. NaB induces the apoptosis of HCT-116cellsThe apoptosis of HCT-116induced by NaB is confirmed by hoechst33342staining, where we observed condensation and fragmentation of chromatin in HCT-116cells with lOmM NaB incubated for24h. To determine the apoptosis of HCT-116at different dose and time of NaB, the apoptosis of HCT-116was quantified by annexin V+PI assay. Compared with the control, the apoptosis of HCT-116increase is correlated with the concentration and time exposure of NaB. This suggests that the increase of apoptosis of HCT-116by NaB is dose and time-dependent. Furthermore, we measure the cleavage of PARP, which is a marker of the apoptotic response, by western blot at different dose and time of NaB. A clear increase in the percentage of cleavage of PARP degradation24h after treatment with10mM NaB was noted, which is in coincident with the apoptosis of HCT-116quantitified by annexin V+PI assay.2. Ca2+is involved in NaB-induced apoptosis of HCT-116cellsTo examine the role of the Ca2+in the apoptosis of HCT-116by NaB, extracellular Ca2+was chelated by0.5mM EGTA. Compare with those of NaB-treated alone, preincubation with EGTA exhibited a significantly attenuation of the apoptosis of HCT-116. Furthermore, chelated the intercellular Ca2+with10μM BAPTA-AM have shown same results. These data suggest that the extracellular Ca2+influx implicated in the apoptosis of the HCT-116induced by NaB.3. SOCE is involved in NaB-induced apoptosis of HCT-116cellsTo identify the SOCE is involved in NaB-induced apoptosis in HCT-116cells, HCT-116was preincubated with5μM SKF96365and75μM2-APB which were the pharmacologic inhibitor of SOCE. The apoptosis of HCT-116was decreased significantly measured by annexin V+PI assay compared with those of the control cells. Meantime the percentage of the cleavage of PARP degradation in the pretreated cells with SKF96365or2-APB decreased compared with the control cells treated by NaB only.In order to confirm the involvment of SOCE in the apoptosis of HCT-116induced by NaB, STIM1, a key molecule of SOCE located mostly in the ER, was first downregulated. Follwing the downrgulation of STIM1, NaB-induced apoptosis was also antenuated. The efficiency of downregulation is determined by real-time PCR and western blot The apoptosis of HCT-116was quantitified by annexin V+PI assay and the percentage of cleavage of PARP degradation decreased significantly after the the downregulation of STIM1.4. The apoptosis of HCT-116induced by NaB are independent on the expression of STIMlTo determine whether the apoptosis of HCT-116induced by NaB is dependent of the transcriptional activation of STIM1, just like the way NaB works on other proapoptosis genes in cancer cell apoptosis, we measured the levels of protein STIM1in HCT-116at different dose and time under NaB treatment. Immunoblotting with anti-STIM1antibody showed no difference in the protein of STIM1in treated cells in comparison to those of the control cells. This result indicated that the role of NaB is independent of the expression of STIM1.5. NaB triggers the translocation and colocalization of STIM1and OrailWe investigated the change of STIM1localization followed by NaB-treatment. The treatment of NaB triggered a redistribution of STIMl into puncta. Moreover, STIM1and Orail were colocalized in the same puncta that have previously been characterized. These results indicate the treatment by NaB induced the translocation of STIM1and Orail, which suggests that the apoptosis induced by NaB is correlated with the redistribution of STIM1and Orail.ConclusionIn conclusion, this study shows for the first time that extracellular Ca2+influx, which is mediated by SOC channels in a STIM1-dependent manner, is involved in sodium butyrate-induced apoptosis in colon cancer cells. Theses data not only provide new insights into the molecular mechanism of the pro-apoptotic action of sodium butyrate in colon cancer cells, but also highlights the role of SOCE in the regulation of apoptosis.更多还原