【作者】 陈小军；

【导师】 顾立刚；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2008， 博士

【摘要】 肿瘤细胞的过度增殖或凋亡不足在肿瘤病理机制中起到重要的作用。转录因子Elk1属于ETS原癌基因家族成员之一,是原癌基因c-fos的调节因子,其磷酸化与原癌基因c-fos的转录激活密切相关,与细胞分化、增殖、凋亡和肿瘤发生发展相关,可能是恶性肿瘤发生发展的重要环节及细胞侵袭转移的调控通路。外界各种刺激经MAPK信号通路磷酸化Elk1,引起Elk1构象改变,从而增强其DNA结合活性,激活转录活性,上调生长分化相关基因,抑制凋亡相关基因,因而已成为肿瘤基因治疗中的一个新靶点。因此,探讨其相关调控机制,将有助于临床应用中医药对肿瘤的基因治疗。中医药在治疗肿瘤上有其特殊优势。单味中药和中药复方具有多种有效成分,奠定了中药多靶点、多环节、多部位效应的物质基础。因此,在中医理论指导下,结合分子生物学现代技术,深入研究恶性肿瘤中医药多靶点联合治疗的机制,将有希望成为肿瘤治疗和抗复发、转移的重要手段。本研究通过建立与细胞增殖调控关系最为密切的Elk1信号转导通路筛选平台,筛选目前临床上已经使用的四种抗肿瘤中药注射液(即艾迪注射液、康莱特注射液、华蟾素注射液、榄香烯注射液)对细胞增殖相关的信号转导通路靶分子Elk1的影响,并对筛选的结果用Western Blot实验进行二次验证,并进一步对榄香烯注射液抑制肿瘤细胞增殖、促进肿瘤细胞凋亡的作用和分子机理进行探讨。1目的建立与细胞增殖相关的Elk1信号转导通路筛选平台;筛选四种抗肿瘤中药注射液(即艾迪注射液、康莱特注射液、华蟾素注射液、榄香烯注射液)对细胞增殖相关的信号转导通路靶分子Elk1的影响;评价榄香烯注射液对肿瘤细胞增殖和凋亡的影响;探讨榄香烯注射液对肿瘤细胞增殖和凋亡影响的作用机理。2方法2.1以人宫颈癌HeLa细胞为模型,选取与细胞增殖调控关系最为密切的Elk1信号传导通路建立筛选平台。首先通过MTT法检测四种中药注射液对HeLa细胞的抑制作用,确定各自的IC50值;然后应用Elk1双萤光素酶报告系统进行筛选,并对筛选的结果用Western Blot实验进行二次验证,检测四种中药注射液对HeLa细胞磷酸化Elk1及其靶基因c-fos表达的影响。2.2以HeLa和HepG2细胞为模型,采用MTT法测定不同浓度的榄香烯注射液对细胞增殖的抑制作用,并确定IC50值;采用光学显微镜和荧光显微镜观察IC50浓度的榄香烯注射液对细胞形态的影响;采用流式细胞分析术,结合荧光染料FITC-Annexin-V和PI对细胞膜磷脂酰丝氨酸的外翻进行分析,观察IC50浓度的榄香烯注射液诱导肿瘤细胞凋亡的作用;采用流式细胞术,结合荧光染料PI对细胞中的DNA含量进行分析,观察IC50浓度的榄香烯注射液对肿瘤细胞增殖的抑制作用;采用Elk1双荧光素酶反式报告基因的筛选技术和Western Blot等技术,观察IC50浓度的榄香烯注射液对Elk1萤光素酶活性、磷酸化的Elk1及其靶基因c-fos的表达的影响,以及对MAPK通路激酶磷酸化、膜受体通路(Caspase-8)和线粒体通路(Caspase-9)活性的影响。3结果3.1抗肿瘤注射液对信号转导分子Elk1活性的筛选3.1.1成功建立与细胞增殖相关的Elk1信号转导通路筛选平台报告质粒pFA- Elk1编码GAL4dbd和Elk1的融合蛋白,pFR-luc编码萤火虫(Firefly)荧光素酶分子,其启动子区含有GAL4dbd的结合位点GAL4UAS,所以萤火虫荧光素酶活性可以反映细胞内转录因子Elk1的活性(磷酸化形式)。内对照质粒pRL-TK编码海肾(Renilla)荧光素酶分子,由TK启动子调控。实验中使用pcDB空载体作为阴性对照,pcDB-MEK1作为激活Elk1的阳性对照。实验结果显示阳性对照分子的活性与预期相符,验证了实验系统的可靠性。3.1.2四种中药注射液对HeLa细胞增殖的抑制作用艾迪注射液100μL/孔作用24h对HeLa细胞体外增殖有明显的抑制作用,求其IC50值为300.60mg·mL-1。康莱特注射液0.39~100μL/孔作用24h对HeLa细胞体外增殖无明显的抑制作用。华蟾素注射液6.25~100μL/孔作用24h对HeLa细胞体外增殖有一定抑制作用,求其IC50值为51.65mg·mL-1。榄香烯注射液1.56~100μL/孔作用24h对HeLa细胞体外增殖有明显的抑制作用,呈剂量依赖性,求其IC50值为0.08mg·mL-1。3.1.3四种中药注射液对HeLa细胞Elk1萤光素酶活性的影响IC50浓度的上述四种中药注射液处理24h后,通过Elk1双萤光素酶报告系统检测发现,与对照组比较,榄香烯处理组Elk1活性明显受到抑制,艾迪、华蟾素处理组Elk1活性有一定的下调,而康莱特处理组无抑制作用。3.1.4四种中药注射液对HeLa细胞磷酸化Elk1及其靶基因c-fos表达的影响磷酸化的Elk1及其靶基因c-fos在HeLa细胞中稳定高表达,以IC50浓度的上述四种中药注射液处理24h后,通过Western Blot检测发现,榄香烯处理组磷酸化的Elk1及其靶基因c-fos的表达明显下调,艾迪、华蟾素处理组磷酸化的Elk1有一定程度下调,c-fos的表达明显下调,而康莱特处理组无显著抑制作用。3.2榄香烯注射液对肿瘤细胞增殖和凋亡影响的作用机理研究3.2.1榄香烯注射液对HeLa细胞、HepG2细胞增殖的抑制作用在第一部分的中药注射液筛选中,结果提示榄香烯注射液1.56~100μL/孔作用24h对HeLa细胞体外增殖有明显的抑制作用,呈剂量依赖性,求其IC50值为0.08mg·mL-1。榄香烯注射液0.39~100μL/孔作用24h对HepG2细胞体外增殖有明显的抑制作用,求其IC50值为0.2mg·mL-1。阳性对照紫杉醇注射液0.39~100μL/孔作用24h对HeLa细胞体外增殖有明显的抑制作用,求其IC50值为0.42mg·mL-1。紫杉醇注射液0.39~100μL/孔作用24h对HepG2细胞体外增殖有明显的抑制作用,求其IC50值为0.9mg·mL-1。3.2.2光学显微镜和荧光显微镜观察HeLa细胞、HepG2细胞经IC50浓度的榄香烯分别作用24h后,经光学显微镜观察出现变小、变圆、皱缩、染色质凝集,并从培养皿脱落等现象,经荧光显微镜观察出现凋亡Ⅰ期的细胞核呈波纹状或呈折缝样,部分染色质出现浓缩状态,Ⅱa期细胞核的染色质高度凝聚、边缘化,Ⅱb期的细胞核裂解为碎块,产生凋亡小体,即死细胞数目增多。这些现象与阳性对照紫杉醇注射液引起的细胞改变相一致,是细胞凋亡的典型特征。而在空对照组细胞状态良好。3.2.3细胞膜磷脂酰丝氨酸外翻分析IC50浓度的榄香烯和紫杉醇注射液分别作用24h后,均可看到细胞FITC-Annexin-V荧光强度增加,说明细胞质膜的PS已经外翻;同时PI荧光强度也有所增加,提示细胞质膜的完整性受到损坏,说明除了凋亡外还存在其它形式的细胞死亡方式。3.2.4细胞周期分析无论是在HeLa细胞还是HepG2细胞,经IC50浓度的榄香烯和紫杉醇注射液分别作用24h后,与空对照相比,榄香烯注射液能阻滞细胞生长于S期和/或G2M期。阳性对照紫杉醇注射液能阻滞细胞生长于G1S期。3.2.5 Elk1信号通路的筛选与空白对照组相比,IC50浓度的榄香烯及紫杉醇注射液处理24h后可抑制Elk1荧光素酶报告活性;而且在PMA+I的刺激下,Elk1荧光素酶报告活性可被榄香烯及紫杉醇注射液抑制,说明二者均能影响HeLa和HepG2细胞Elk1转录活性。3.2.6 Western Blot检测磷酸化Elk1及其靶基因c-fos的表达以β-actin为内对照,与空对照比较,榄香烯注射液能抑制HeLa和HepG2细胞中磷酸化Elk1的水平以及c-fos的表达,而阳性对照紫杉醇注射液只能抑制HepG2细胞中磷酸化Elk1的水平以及c-fos的表达,对HeLa细胞中磷酸化Elk1的水平以及c-fos的表达无显著影响。3.2.7 MAPK通路激酶磷酸化的Western印迹分析用抗磷酸化ERK抗体和抗非磷酸化ERK抗体检测,与空对照组比较,除阳性对照紫杉醇组能显著抑制HeLa细胞和HepG2细胞中磷酸化ERK的水平以外,榄香烯组磷酸化ERK的水平均无明显差异。用抗磷酸化JNK抗体和抗非磷酸化JNK抗体检测,与空对照组比较,榄香烯及紫杉醇注射液均能显著抑制HeLa细胞和HepG2细胞中磷酸化JNK的水平,磷酸化JNK的比例显著降低。用抗磷酸化p38抗体和抗非磷酸化p38抗体检测,与空对照组比较,除阳性对照紫杉醇组能显著抑制HeLa细胞中磷酸化p38水平以外,其余各组磷酸化p38水平均无明显差异。3.2.8 Western印迹检测榄香烯对caspase活性的影响用抗caspase-8抗体和抗caspase-9抗体检测,与空对照组比较,在HepG2细胞中,榄香烯和紫杉醇组cleaved caspase-8和caspase-9的水平都有显著提高;在HeLa细胞中,榄香烯和紫杉醇组cleaved caspase-8的水平有显著提高,full length caspase-9的比例均明显降低,而cleaved caspase-9的水平无明显差异。4结论4.1成功建立了与细胞增殖相关的Elk1信号转导通路筛选平台。4.2四种中药注射液对肿瘤细胞具有选择性抑制作用,且呈剂量依赖性。榄香烯注射液对Hela细胞体外增殖有明显的抑制作用,艾迪、华蟾素注射液有一定程度的抑制作用,而康莱特注射液无明显抑制作用。4.3榄香烯、艾迪、华蟾素注射液抑制人宫颈癌HeLa细胞生长的可能机制之一是下调了Elk1转录活性、转录因子Elk1的磷酸化水平及其靶基因c-fos的表达。康莱特注射液对Elk1萤光素酶活性、磷酸化的Elk1及其靶基因c-fos的表达无显著抑制作用,提示是否还有其它的作用机制尚须进一步探讨。4.4榄香烯注射液能显著抑制HeLa和HepG2细胞增殖,且呈剂量依赖性,其IC50值分别为0.08mg·mL-1和0.2mg·mL-1;阻滞HeLa细胞于S期,HepG2细胞于S和G2M期;能诱导HeLa细胞出现早期凋亡现象,HepG2细胞出现早期和中晚期凋亡现象。4.5榄香烯注射液通过调控JNK MAPK通路,抑制Elk1转录活性,下调转录因子Elk1的磷酸化水平及其靶基因c-fos的表达,发挥抑制肿瘤细胞增殖的作用;通过调节膜受体通路(Caspase-8)和/或线粒体通路(Caspase-9),从而发挥促进肿瘤细胞凋亡的生物学功能。综上所述,本研究筛选了中药注射液对细胞增殖相关的信号传导通路靶分子Elk1的影响,发现榄香烯注射液的作用尤为显著。证实了榄香烯抑制肿瘤细胞增殖、促进肿瘤细胞凋亡的功能,其分子机理是通过调控JNK MAPK通路,抑制Elk1转录活性,下调转录因子Elk1的磷酸化水平及其靶基因c-fos的表达,调节膜受体通路(Caspase-8)和/或线粒体通路(Caspase-9),从而发挥抑制肿瘤细胞增殖、促进肿瘤细胞凋亡的生物学功能。这些工作为研究中药注射液的功能和分子机理的线索,并为临床用药提供参考,对于推动中医药抗肿瘤作用机制的深入研究具有十分重要的意义。更多还原

【Abstract】 Dysfunction of proliferation and apoptosis in human cancer cells is most important during the pathogenesis of the cancer. The transcription factor Elk1 is one of the Ets family members, and identified as a regulator of proto-oncogene c-fos. Phosphorylation of Elk1 regulates the transcriptional activation of c-fos, which is related with cell differentiation, proliferation, apoptosis and oncogenesis. Phosphorylation of Elk1 also regulates the progression and metasis of cancer. Elk1 is phosphorylated by the extracellular stimulation through MAP kinases, which triggers the conformational change to enhanced DNA binding, stimulation of its transcriptional activation. Elk1 becomes a new molecular target in the cancer gene therapy. So, investigation of Elk1 regulational mechanism will be helpful to clinical application of TCM in cancer patients.TCM plays a special important role in cancer therapy. The simple medica and the compound recipe have many effective components, which is the material basis of TCM multiple target. So, under the principle of TCM theory, combining the modern molecular biology techniques, to investigate the mechanism of TCM multiple target is most important in cancer therapy. This experiment established the cell proliferation related Elk1 signal transduction pathway screening system based on dual-luciferase reporter assay to screen the clinical applied four kinds of TCM anti-tumor injections (such as AiDi injection, KangLaiTe injection, Cinobutacini injection and Elemene injection), and further investigated the effect and mechanism of Elemene injection on HeLa and HepG2 cells.1 ObjectiveTo establish the cell proliferation related Elk1 signal transduction pathway screening system based on dual-luciferase reporter assay; To screen the clinical applied four kinds of TCM anti-tumor injections (such as AiDi injection, KangLaiTe injection, Cinobutacini injection and Elemene injection) in Elk1 signaling pathway; To observe the effect of Elemene injection on HeLa and HepG2 cells; To investigate the mechanism of Elemene injection on HeLa and HepG2 cell proliferation and apoptosis.2 Methods2.1 In human cervical cancer HeLa cell,we established the cell proliferation related Elk1 signal transduction pathway screening system based on dual-luciferase reporter assay. MTT assay was used to identify the potential inhibitory activity of four kinds of TCM injections, and the individual 50 % inhibitory concentration (IC50) was decided. Elk1 screening system was applied to screen the effect of four kinds of TCM injections in Elk1 signaling pathway, which was evidenced by Western Blot analysis on expression of phospho-Elk-1 and its target gene c-fos.2.2 In human HeLa and HepG2 cell, MTT assay was used to observe the antiproliferation effect of Elemene injection, and the IC50 was decided. The light and fluorescent microscopy were used to observe the effect of Elemene injection on cell morphological changes. Cell cycle and apoptosis were analysized by flow cytometry. Elk1 screening system and Western Blot analysis were applied to investigate the effect of Elemene injection in Elk1 transcriptional activity, the expression of phospho-Elk-1 and its target gene c-fos, phosphorylation of MAP kinases, caspase-8 and caspase-9 activities.3 Results3.1 Screening of TCM anti-tumor injections inhibitor of Elk1 acticity3.1.1 Established the cell proliferation related Elk1 signal transduction pathway screening system successfullyReporter plasmid pFA- Elk1 encodes GAL4dbd and Elk1 fusion protein, pFR-luc encodes firefly luciferase, in which promoter there is GAL4dbd domain, so firefly luciferase activity can indicate the cell transcription factor Elk1 activity. The internal compared plasmid pRL-TK encodes Renilla luciferase, which is controlled by TK promoter. The pcDB was negative control, and pcDB-MEK1 was positive control. We found the result was in accordance with what we thought, so the Elk1 screening system is confidenced.3.1.2 Inhibitory activity of four kinds of TCM injections on HeLa cell proliferationAiDi injection 100μl/well for 24h inhibited the HeLa cell proliferation, and its IC50 is 300.60mg/ml。KangLaiTe injection 0.39~100μl/well for 24h can not inhibit the HeLa cell proliferation. Cinobutacini injection 6.25~100μl/well for 24h inhibited the HeLa cell proliferation, and its IC50 is 51.65mg/ml. Elemene injection 1.56~100μl/well for 24h inhibited the HeLa cell proliferation, and its IC50 is 0.08mg/ml.3.1.3 Effect of four kinds of TCM injections on Elk1 transcriptional activity After treatment of IC50 concentration four kinds of TCM injections respectively for 24h in HeLa cell, Elk1 transcriptional activity was obviously inhibited by the Elemene injection, as well as AiDi injection and Cinobutacini injection, while KangLaiTe injection had no inhibitory effect.3.1.4 Effect of four kinds of TCM injections on the expression of phospho-Elk-1 and its target gene c-fos The expression of phospho-Elk-1 and its target gene c-fos were high in HeLa cell. After treatment of IC50 concentration four kinds of TCM injections respectively for 24h in HeLa cell, the expression of phospho-Elk-1 and its target gene c-fos were down-regulated, as well as AiDi injection and Cinobutacini injection, while KangLaiTe injection had no obviously inhibitory effect.3.2 Investigation of the effect and mechanism of Elemene injection on proliferation and apoptosis3.2.1 The antiproliferation effect of Elemene injection on human HeLa and HepG2 cellThe first part showed that Elemene injection 1.56~100μl/well for 24h inhibited the HeLa cell proliferation, and its IC50 is 0.08mg/ml. Elemene injection 0.39~100μl/ well for 24h inhibited the HepG2 cell proliferation, and its IC50 is 0.2mg/ml. The positive control Paclitaxel injection 0.39~100μl/well for 24h inhibited the HeLa cell proliferation, and its IC50 is 0.42mg/ml. Paclitaxel injection 0.39~100μl/well for 24h inhibited the HepG2 cell proliferation, and its IC50 is 0.9mg/ml。3.2.2 The light and fluorescent microscopy observationAfter treatment of IC50 concentration Elemene injection for 24h respectively in HeLa and HepG2 cell, there were some signs of classic apoptotic changes, such as cell shrinkage, marked rounding, condensation of chromatin at the nuclear periphery and eventually detached from the culture wells. Meanwhile, we used the fluorescent dye—DAPI to validate the effect on apoptosis. This result is in accordance with the light microscopy findings, identifying the elemene related to apoptosis.3.2.3 Apoptosis analysis by flow cytometryThe exposure of phosphatidylserine on the cell membrane is readily detected by Annexin V staining. Thus PI and Annexin V-FITC double staining should detect late apoptosis/necrosis whereas single PI staining should detect dead cells. we detected PS surface exposure by FITC/annexin-V staining of cells 24 h after treatment of IC50 Elemene injection. Plasma membrane integrity was simultaneously assessed by PI dye exclusion using two-color fluorescence-activated cell sorting (FACS) analysis. In HeLa and HepG2 cells treated with the elemene and paclitaxel respectively, the proportion of Annexin-V- and/or PI-positive cells was evidently increased compared with mock cells. These findings indicated that these cells underwent both necrosis and apoptosis.3.2.4 Cell cycle analysis by flow cytometryIn cell cycle analysis, the cells in S and G2/M phase represent the proliferating cells. After treatment of IC50 concentration Elemene injection for 24h respectively in HeLa and HepG2 cell, the percent of G2/M and S phase were decreased, which indicated there was a significant inhibitory effect on cell growth.3.2.5 Elk1 screening system In a dual-luciferase reporter assay system, the Elk-1 reporter activity was measured after incubation with the IC50 concentration Elemene injection for 24 h respectively in HeLa and HepG2 cell. Each experiment was performed in triplicate and each assay was repeated at least three times. We found that elemene down-regulated the Elk-1 transcriptional activity compared with the mock even if there is a stimulus (PMA+I).3.2.6 Western Blot analysis of the expression of phospho-Elk-1 and its target gene c-fosCompared to the untreated cells, phosph-Elk-1 and its target gene c-fos expression levels in HeLa and HepG2 cell were decreased by Elemene injection, in agreement with dual-luciferase assay results, as well as the positive control Paclitaxel injection in the HepG2 cell,while no inhibitory effect in HeLa cell.3.2.7 Western Blot analysis of MAP kinases phosphorylationCompared to the untreated cells, the treatment of elemene significantly decreased JNK activity, but did not influence activities of ERK and P38, compared to the untreated cells, which confirmed that elemene modulated JNK kinases activity, but not ERK or p38 MAPK.3.2.8 Western Blot analysis of caspase-8 and caspase-9 activitiesCompared to the untreated cells, the treatment of Elemene and Paclitaxel injection significantly increased cleaved caspase-8 and caspase-9 levels in HepG2 cell, while in HeLa cell significantly increased the cleaved caspase-8, decreased full length caspase-9 but did not influence the level of cleaved caspase-9.4 Conclusion4.1 Established the cell proliferation related Elk1 signal transduction pathway screening system successfully.4.2 Elemene injection can inhibit human cervix cancer HeLa cell proliferation, as well as AiDi and Cinobutacini injection, while KangLaiTe has no inhibition.4.3 Elemene, AiDi and Cinobutacini injection can inhibit human cervix cancer HeLa cell proliferation, which may be related with suppression of c-fos gene through inhibiting expression of phosphorated Elk1, while KangLaiTe has no inhibition.4.4 Elemene injection inhibited HeLa and HepG2 cell growth in dose-dependent manner, its IC50 is 0.08mg/ml and 0.2mg/ml respectively; blocked the HeLa cell cycle progression in S phase, HepG2 cell cycle progression in S and G2M phase; inducted the early apoptosis in HeLa cell, the early and middle late apoptosis in HepG2 cell.4.5 Elemene injection down-regulated Elk-1 transcriptional activity, decreased the phosphorylated Elk-1 and its target gene c-fos expression level through modulating the JNK MAPK pathway, leading the inhibition of cell proliferation; inducted the apoptosis through modulating the caspase-8 and caspase-9. To sum up, TCM anti-tumor injections (such as AiDi injection, Cinobutacini injection and Elemene injection) can inhibit the cell proliferation related Elk1 signal transduction pathway, especially the Elemene injection. Elemene injection has the effect of inhibition of cell proliferation and induction of apoptosis, and its molecular mechanism may be down-regulation of Elk-1 transcriptional activity and its target gene c-fos expression levels by by modulating the JNK pathway, and may be also modulation of caspase-8 and caspase-9.更多还原