【作者】 刘卫海；

【导师】 赖小平；

【作者基本信息】 广州中医药大学 ， 中医学， 2011， 博士

【摘要】 研究背景与目的中药藤黄(Gamboge)系藤黄科植物藤黄树(Garcinia hanburyi Hook. F. G)所分泌出的干燥树脂,近年来,其抗肿瘤作用受到高度关注。已经有研究表明,新藤黄酸(neogambogic acid. NGA)为藤黄抗肿瘤作用的主要有效成分之一,有望开发成抗肿瘤新药。但目前对其抗瘤谱的筛选尚不完全,尚未查阅到给药后新藤黄酸在体内分布的资料,对其抗肿瘤作用机制的研究也仅限于新藤黄酸对白血病细胞周期的影响,许多对该药物的应用较为重要的资料仍不完善。鉴于此,本文旨在进一步探讨新藤黄酸的体外和体内抗肿瘤活性,并在此基础上从细胞凋亡、细胞周期、细胞信号转导、端粒酶活性以及血管生成等角度入手,探索新藤黄酸的抗肿瘤作用机制,为新藤黄酸的开发及临床应用奠定理论基础。方法与结果1新藤黄酸的体外抗肿瘤作用运用MTT法和CCK-8法,通过测定新藤黄酸对体外培养肿瘤细胞的抑制率求出新藤黄酸对各肿瘤细胞株的IC50(半数抑制浓度),考察新藤黄酸对体外培养肿瘤细胞的抗增殖作用,结果发现新藤黄酸对非选择性获得的16株肿瘤细胞均具有抗增殖作用,IC50在1.14～4.25μg/mL之间,说明新藤黄酸具有确切的体外抗肿瘤活性(IC50<10μg/mL)。对人正常肝细胞株HL-7702的抗增殖实验结果显示,新藤黄酸对人正常肝细胞HL-7702的IC50为5.23±0.04μg/mL,新藤黄酸对人肝细胞癌Hep G2细胞株的IC50为1.22±0.12μg/mL,提示新藤黄酸对正常肝细胞的增殖有一定的抑制作用,但作用强度较其对肿瘤细胞的作用强度弱,推论在一定浓度范围内(1.25-2.50μg/mL)新藤黄酸具有选择性抑制肿瘤细胞增殖的作用。2新藤黄酸的体内抗肿瘤作用经过急性毒性试验,运用SPSS11.5统计学软件按机率单位加权回归法(Bliss法)计算得出新藤黄酸对小鼠的LD50(半数致死量)为36.66mg/kg,在此基础上确定小鼠实验的高、中、低三个剂量分别为8.0 mg/kg、4.0 mg/kg、2.0 mg/kg.采用荷S180腹水瘤小鼠模型,以存活时间为指标,观察新藤黄酸对动物肿瘤的体内抗肿瘤作用。结果显示,NGA中剂量(4.0 mg/kg)对延长荷瘤小鼠的存活时间效果最好,明显高于阳性对照药氟尿嘧啶(5-FU) (10.0 mg/kg)的疗效(P<0.05),但NGA高剂量(8.0 mg/kg)和NGA低剂量(2.0 mg/kg)的疗效均低于5-FU (10.0 mg/kg)的疗效(P<0.05),且NGA低剂量的平均存活时间与阴性对照组的差异无统计学意义(P>0.05)。提示新藤黄酸发挥治疗作用的剂量范围较窄,高剂量组的疗效比中剂量组差可能是由新藤黄酸的毒副作用所引起的。在建立小鼠血液及器官组织中新藤黄酸的HPLC测定方法后,建立荷S180实体瘤模型,考察给药后新藤黄酸在小鼠体内的分布情况。结果显示,腹腔注射后新藤黄酸在心、肝、肺、脾、肾及肿瘤组织中的峰浓度值分别为9.58±2.79、16.49±4.17、14.51±3.68、8.23±1.88、8.85±2.37、10.28±2.75μg/g,但在试验条件下,未能在脑组织中检测到新藤黄酸,其原因可能是新藤黄酸未能通过血脑屏障或者浓度水平未达到检测限。根据体内分布实验结果和体外实验结果,确定裸小鼠移植瘤模型的瘤株为人肝细胞癌Hep G2细胞株,建立人肝细胞癌Hep G2裸小鼠移植瘤模型,以相对肿瘤体积(relative tumor volume, RTV)和抑瘤率(inhibition rate of tumor growth, TGI)为指标考察新藤黄酸对人肿瘤的体内抗肿瘤作用,结果显示,新藤黄酸对人肝细胞癌Hep G2裸小鼠移植瘤的生长具有明显的抑制作用,在实验时间范围内,给药8d后,可观察到RTV随着给药剂量的增大而减小,给药14d后,阴性对照组、5-FU组、NGA高剂量(8.0 mg/kg)组、中剂量(4.0 mg/kg)组和低剂量(2.0 mg/kg)组的RTV分别为10.72±4.83、7.57±2.24、1.34±0.36、2.52±1.37和7.02±1.94；5-FU组、NGA高剂量(8.0 mg/kg)组、中剂量(4.0 mg/kg)组和低剂量(2.0 mg/kg)组的TGl分别为46.54%、83.75%、68.09%和22.14%；提示在一定剂量范围内,新藤黄酸具有确切的体内抗肿瘤作用。3新藤黄酸的抗肿瘤作用机制在确定新藤黄酸具有确切的抗肿瘤作用后,从细胞凋亡、细胞周期、细胞信号转导、端粒酶活性和肿瘤血管生成五个方面进一步研究新藤黄酸的抗肿瘤作用机制。采用Annexin V-FITC/PI双染流式细胞术检测新藤黄酸干预后体外培养HepG2细胞的早期凋亡率,结果显示,早期凋亡率的最大值为(49.44±3.12)%,且出现在给药后24h,说明新藤黄酸具有诱导体外培养Hep G2细胞早期凋亡的作用。琼脂糖凝胶电泳DNA片段化分析的结果显示,新藤黄酸能使Hep G2细胞核DNA发生明显的降解,但未观察到细胞凋亡的特征DNA梯状条带,其原因可能是细胞在发生早期凋亡后,DNA又被进一步降解成200bp左右的片段。为了进一步研究细胞凋亡的机制,采用Western blot法检测给药后Hep G2细胞中凋亡相关蛋白Bax、Bcl-2的水平,结果显示,Bax呈浓度依赖性升高,Bcl-2呈浓度依赖性降低；对照组、NGA(1.0μg/mL)组、NGA(2.Oμg/mL)组和NGA(3.0μg/mL)的Bax/Bcl-2比值分别为0.06、0.29、2.25和21.14。说明新藤黄酸在一定浓度范围内可以上调体外培养Hep G2细胞的Bax/Bcl-2比值。免疫组化法检测人肝细胞癌Hep G2裸小鼠移植瘤组织Bax、Bcl-2蛋白水平的结果显示,NGA低剂量(2.0mg/kg)组的Bax蛋白水平和Bcl-2蛋白水平与阴性对照组相比无显著差异(P>0.05),NGA中剂量(4.0mg/kg)组和NGA高剂量(8.0mg/kg)组的Bax蛋白水平明显高于阴性对照组,而Bcl-2蛋白水平明显低于阴性对照组；阴性对照组、NGA(2.0mg/kg)组、NGA(4.0mg/kg)组和NGA(8.0mg/kg)的Bax/Bcl-2比值分别为0.729、0.808、1.679和1.688。提示新藤黄酸在一定剂量范围内可以上调Hep G2裸小鼠移植瘤组织的Bax/Bcl-2比值。可见,体外试验结果和体内试验结果均支持新藤黄酸可通过上调Bax的水平和(或)下调Bcl-2的水平,从而升高Bax/Bcl-2比值诱导细胞凋亡。在对细胞周期进展的影响方面,采用PI单染流式细胞术检测处于各细胞周期时相的细胞数占总细胞数的百分比,结果显示,与对照组相比,给药后12～36h,S期细胞明显增多,在给药后48h,G0/G1期细胞增多,说明新藤黄酸能使细胞阻滞在S期,且随着时间的延长,能进一步使细胞阻滞在G0/G1期。在对MAPK细胞信号转导通路的影响方面,采用免疫组化法检测Ras/Raf/MEK/ERK级联通路中ERK和MEK的活化形式p-ERK1/2和p-MEK1/2的水平,结果显示,新藤黄酸可下调ERK1/2蛋白和MEK1/2蛋白的磷酸化水平,说明新藤黄酸可以通过下调ERK信号通路的信号引起细胞周期阻滞从而抑制细胞的增殖。但其调节点可能在Ras/Raf/MEK/ERK级联通路中MEK的上游,因为MEK的活化形式p-MEK1/2的水平也得到了下调。在对端粒酶活性的影响方面,采用荧光定量PCR法,检测端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)的mRNA水平来间接反映端粒酶的活性。结果显示,新藤黄酸给药各组Hep G2细胞的hTERT mRNA水平都明显低于对照组(P<0.05),且新藤黄酸给药各组的Hep G2细胞hTERT mRNA水平随给药浓度的升高而降低,各组间均有显著差异(P<0.05)。说明新藤黄酸对端粒酶活性具有抑制作用,且表现为浓度依赖性增强。据此推断,新藤黄酸可通过抑制端粒酶活性发挥体外抗肿瘤作用。在对肿瘤细胞血管生成的影响方面,运用免疫组化法检测Hep G2裸小鼠移植瘤组织中CD31(血小板内皮细胞粘附分子)的水平,从而反映新藤黄酸对肿瘤血管生成的影响,实验结果显示,新藤黄酸3个给药组的CD31阳性表达量IOD值均明显低于阴性对照组,且差异具有统计学意义(P<0.05),提示在一定浓度范围内,新藤黄酸可降低实体瘤组织的CD31水平,说明新藤黄酸对实体肿瘤的血管生成有一定的抑制作用。结论新藤黄酸具有广谱的体外抗肿瘤作用,在一定浓度范围内,新藤黄酸可选择性抑制肿瘤细胞的增殖,而对正常细胞的抗增殖作用相对较弱。新藤黄酸给药后能在小鼠体内广泛分布,其中以肝脏分布最高,在肿瘤组织中也有较高的分布；新藤黄酸可显著延长荷S180腹水瘤小鼠的存活时间,说明新藤黄酸对动物肿瘤具有确切的体内抗肿瘤活性；新藤黄酸能显著减小人肝细胞癌Hep G2裸小鼠移植瘤的相对肿瘤体积,抑瘤率可达到83.75%,说明新藤黄酸对人肝细胞癌Hep G2裸小鼠移植瘤具有确切的体内抗肿瘤活性。新藤黄酸可通过诱导细胞凋亡、阻滞细胞周期进展、抑制肿瘤血管生成而发挥抗肿瘤作用。新藤黄酸诱导肿瘤细胞凋亡与其对上调Bax/Bcl-2的比值有关。另外,新藤黄酸尚可通过下调MAPK/ERK信号通路关键酶的活性阻断MAPK级联反应,从而引起细胞周期阻滞,产生抗增殖作用；同时,细胞周期阻滞尚可诱导肿瘤细胞程序性死亡。更多还原

【Abstract】 Background and purposeGamboge is the dry resin secreted from Garcinia hanburyi Hook.F.G. In recent years, the anti-tumor effect of Gamboge has been gradually and greatly concerned. Studies have confirmed that neogambogic acid (NGA) is one of the main active ingredients of the anti-tumor effect of Gamboge and that it is expected to be developed into a new anti-tumor drug. But the research on its anti-tumor spectrum has not yet completed. It has not been found that the information of distribution of NGA in the organs and tissues. In addition, the research on its mechanism was limited to NGA impacting on cell cycle of leukemia. Much more important information concerning the application of NGA was far from perfect. In view of this, the dissertation aims to further elaborate the anti-tumor activity of NGA in vitro and in vivo. And on this basis, from apoptosis, cell cycle, cell signal transduction, telomerase activity and angiogenesis, we tried to explore the anti-tumor mechanism of NGA and its molecular basis, which would establish the theoretical basis for the development and clinical application of NGA.Methods and results1 The anti-tumor effect of NGA in vitroMTT and CCK-8 were used to assay the inhibition rate of tumor cells treated with NGA in different concentration. According to the inhibition rate, the IC50 (50 percent inhibitory concentration) of each tumor cell line was calculated, which was used to evaluate the anti-proliferative effect of NGA on tumor cells in vitro. The results showed that NGA could play anti-proliferative effect on 16 tumor cell lines. Their IC50 was between 1.14μg/mL and 4.25μg/mL, indicating that NGA has exact anti-tumor activity in vitro (IC50<10μg/mL). The experimental results of the normal human liver cell line HL-7702 treated with NGA showed that the IC50 of NGA on HL-7702 cell line was 5.23±0.04μg/mL, and the IC50 of NGA on human hepatoblastoma Hep G2 cell line was 1.22±0.12μg/mL. In addiction, the results also showed that in a certain concentration range (between 1.25μg/mL and 2.50μg/mL) NGA could selectively play strong anti-proliferative effect on tumor cells while play weaker anti-proliferative effect on normal liver cells.2 The anti-tumor effect of NGA in vivoAfter acute toxicity test, we calculated the LD50 (median lethal dose) of NGA on mice by statistical software SPSS11.5. The result showed that the LD50 was 36.66mg/kg. On this basis, we determined that the high, medium and low doses of the mice experiments were 8.0mg/kg,4.0mg/kg and 2.0mg/kg respectively. The anti-tumor effect of NGA on mice bearing S180 ascitic tumor was evaluated by survival time of tumor-bearing mice. The results showed that NGA at medium dose (4.0 mg/kg) has the best effect on prolonging survival time of tumor-bearing mice, which was significantly higher than the efficacy of the positive control drug 5-FU (fluorouracil)(10.0 mg/kg)(P<0.05). But the efficacies of the high dose (8.0 mg/kg) and low dose (2.0 mg/kg) were lower than the efficacy of 5-FU (10.0 mg/kg) (P<0.05). And the average survival time of low dose group and negative control group had no significant difference (P>0.05). These suggested that NGA played therapeutic effect in a narrow dosage range, and that the efficacy of high dose group was worse than the efficacy of medium dose group may be caused by the toxic side effects of NGA.After establishing the high performance liquid chromatography (HPLC) determination method of NGA in blood and the tissues of organs, the bearing S180 solid tumor model was established to study the distribution of NGA in mice. The results showed that the peak concentration of NGA in heart, liver, lung, spleen, kidney and tumor tissues were 9.58±2.79、16.49±4.17、14.51±3.68.8.23±1.88、8.85±2.37、10.28±2.75μg/g respectively. But in the experimental conditions, NGA failed to be detected in brain tissue, whose reason may be that NGA failed to pass the blood-brain barrier or its concentration level was lower than the detection limit. According to the results of distribution experiment and the results of the experiments in vitro, human hepatoblastoma cell line Hep G2 was determined to be the cell line of the tumor xenograft model in nude mice. Then human hepatoblastoma Hep G2 cell xenograft model in nude mice was established. And the anti-tumor effect of NGA on human tumor in vivo was evaluated by the relative tumor volume (RTV) and tumor growth inhibition rate (TGI). The results showed that the NGA could significantly inhibit the growth of the human hepatoblastoma xenograft in nude mice. In the experimental time frame, after 8 days of treatment, it could be observed that RTV decreased along with the increase of dose. After 14 days of treatment, RTV of the negative control group,5-FU group, NGA high dose (8.0mg/kg) group, medium dose (4.0mg/kg) group and low dose (2.0mg/kg) group were 10.72±4.83,7.57±2.24,1.34±0.36,2.52±1.37 and 7.02±1.94 respectively. TGI of 5-FU group, NGA high dose (8.0mg/kg) group, medium dose (4.0mg/kg) group and low dose (2.0mg/kg) group were 46.54%,83.75%,68.09% and 22.14% respectively. These suggested that in a certain dosage range, NGA had exact anti-tumor effect in vivo.3 The anti-tumor mechanism of NGAAfter determining NGA having the exact anti-tumor effects in vivo and in vitro, we exerted further research on the anti-tumor mechanism of NGA from apoptosis, cell cycle, cell signal transduction, telomerase activity and angiogenesis.After cells stained by Annexin V-FITC/PI, flow cytometry analysis showed that the maximum percentage of apoptosis appeared and reach to (49.44±3.12)% at 24 hours after Hep G2 cells were exposed to 2.0μg/mL NGA, indicating that NGA could induce early apoptosis of Hep G2 cells in vitro. Analysis by agarose gel electrophoresis showed that NGA could induce the DNA in nucleus of Hep G2 evidently to be degraded, but no DNA ladder was observed. Its reason might be that after cells occurring early apoptosis, DNA was further degraded into fragments of about 200 base pairs (bp).To further research on the mechanism of apoptosis, the levels of apoptosis-related proteins Bax and Bcl-2 in Hep G2 cells exposed to NGA in vitro were detected by Western blot. The results showed that Bax increased along with the increase of drug concentration while Bcl-2 decreased along with the increase of drug concentration. The Bax/Bcl-2 ratios of the control group, NGA (1.0μg/mL) group, NGA (2.0μg/mL) group and NGA (3.0μg/mL) group were 0.06,0.29,2.25 and 21.14 respectively, indicating that NGA in a certain concentration range could increase the Bax/Bcl-2 ratio of Hep G2 cells in vitro.The levels of Bax and Bcl-2 in human hepatoblastoma Hep G2 cell xenograft in nude mice was assayed by immunohistochemistry. The results showed that the levels of Bax and Bcl-2 of NGA low dose (2.0mg/kg) group and that of negative control group had no significant difference (P>0.05). For NGA medium dose (4.0mg/kg) group and NGA high dose (8.0mg/kg) group, the levels of Bax were significantly higher than the negative control group, while the levels of Bcl-2 were significantly lower than the negative control group. The Bax/Bcl-2 ratios of the negative control group, NGA (2.0mg/kg) group, NGA (4.0mg/kg) group and NGA (8.0mg/kg) group were 0.729,0.808,1.679 and 1.688 respectively, indicating that NGA in a certain dosage range could increase Bax/Bcl-2 ratio of human hepatoblastoma Hep G2 cell xenograft in nude mice. Evidently, both the results in vitro and in vivo supported the hypothesis that NGA could increase the level of Bax and (or) reduced the level of Bcl-2, which increased Bax/Bcl-2 ratio to induce apoptosis.In terms of the progression of the cell cycle, Hep G2 cells treated with 2.0μg/mL NGA for different time were collected and assayed by flow cytometry. Cell phase analysis showed that compared with the control group, the indexes of the cells arrested in S phase significantly increased during the first 0-36 hours and the indexes of the cells arrested in G0/G1 phase significantly increased when treated with NGA for 48 hours, indicating that NGA could induce cells to be arrested in S phase and to be further arrested in G0/G1 phase with the time passing by.In terms of the MAPK signal transduction pathway, the levels p-ERK1/2 and p-MEK1/2, the activated forms of MEK and ERK of Ras\Raf\MEK\ERK cascade pathway, were assayed by immunohistochemistry. The results showed that NGA could down-regulate the levels of p-ERK1/2 and p-MEKl/2, indicating that NGA could play anti-proliferative effect by reducing the signals of ERK signaling pathway to arrest cell cycle. But the adjustment point may be in the upstream of MEK in Ras/Raf/MEK/ERK cascade pathway, as the level of p-MEK1/2, the MEK activation form, had also been reduced.In terms of telomerase activity, the level of hTERT (human telomerase reverse transcriptase) mRNA was assayed by FQ-PCR (fluorescence quantitative polymerase chain reaction) to reflect telomerase activity. The results showed that the levels of hTERT mRNA of each group of Hep G2 cells treated with NGA was significantly lower than the control group (P<0.05). The level of hTERT mRNA of each group of Hep G2 cells treated with NGA decreased along with the increase of drug concentration, and there is significantly difference among each group (P<0.05). These indicated that NGA could inhibit telomerase activity in drug concentration dependently. Consequently, we inferred that NGA could play anti-tumor effect by inhibiting telomerase activity.In terms of the angiogenesis of tumor, the level of CD31 (platelet endothelial cell adhesion molecule) in Hep G2 tumor tissue in nude mice was assayed by immunohistochemistry. The results showed that CD31-positive expression IOD (integrated option density) values of three groups treated with NGA were significantly lower then that of the negative control group, and the differences were statistically significant (P<0.05). These indicated that NGA could reduce the levels of CD31 in solid tumor tissue in a certain dosage range, suggesting that NGA could inhabit angiogenesis in solid tumors to some extent.ConclusionsNGA has a broad spectrum anti-tumor effect in vitro, In a certain concentration range, NGA can selectively play strong anti-proliferative effect on tumor cells and relatively weaker anti-proliferative effect on normal cells.NGA can be widely distributed in mice. The maximum concentration was found in liver followed by that of the lung. NGA can significantly prolong the life span of the S180 ascites tumor bearing mice, indicating that NGA has an exact anti-tumor activity on animal tumors in vivo. NGA can significantly reduce the RTV of human hepatoblastoma Hep G2 xenografts in nude mice and increase TGI to 83.75%, indicating that NGA has an exact anti-tumor activity on human hepatoblastoma Hep G2 xenografts in nude mice in vivo.NGA can play an anti-tumor effect by inducing apoptosis, arresting cell cycle, inhibiting telomerase activity and inhibiting angiogenesis. NGA inducing tumor cells apoptosis is by increasing the ratio of Bax/Bcl-2. In addition, NGA can blocks MAPK cascade by reducing the activity of key enzymes in MAPK/ERK signaling pathway, causing cell cycle arrest and resulting in anti-proliferative effect. Meanwhile, cell cycle arrest can still induce tumor cell programmed cell death.更多还原