色胺酮对人白血病细胞株K562的体内外抑制作用及其机制研究
|
摘要
1研究目的
色胺酮(Tryptanthrin,TRYP),为吲哚喹唑啉类生物碱,化学名称为吲哚[2,1-b]喹唑啉-6,12-二酮。黄色针晶,熔点267-268°C。最初由马蓝(Strobilanthes cusia)、蓼蓝(Polygonum tinctorium Lour)、菘蓝(Isatis tinctoria L.)等产蓝植物中得到。文献报道TRYP具有抗细菌真菌、抗炎、抗肿瘤及抗寄生虫等多种药理活性。但由于TRYP的来源有限,目前国外多集中在TRYP的分离、提取及合成,药理活性研究多集中在抗炎及少量抗肿瘤等方面。TRYP对血液系统肿瘤的作用也仅局限于对急性早幼粒白血病细胞的影响,但TRYP对慢性髓系白血病细胞增殖、凋亡、细胞周期等作用的研究尚未见报道。本课题以仿生合成得到的TRYP为研究试药,采用体外培养肿瘤细胞和建立体内动物模型等方法,探讨和揭示TRYP对人白血病细胞K562的体内外抑制作用及其机制研究,为TRYP的新药开发提供实验依据。
2方法
2.1 TRYP对8株人源性肿瘤细胞的MTT及抑制K562细胞增殖和诱导凋亡的研究:以A549、HepG2、SW480、Eca-109、GRC-1、LNCAP、A375、K562等8株人源性肿瘤细胞为靶细胞,以抑制增殖为指标,筛选TRYP对肿瘤细胞株的敏感性,然后以最敏感的肿瘤细胞为研究对象;同时,确定TRYP对该敏感细胞体外作用的药物浓度及作用时间;然后取对数生长期的细胞,分别以不同浓度TRYP作用一定时间后,收集细胞,分别采用相差显微镜、Hoechst 33258荧光染色法、透射电镜等方法观察细胞的形态学改变,Annexin-V- FITC和PI双标记流式细胞术检测细胞凋亡率;PI单染色检测细胞周期的变化。
2.2 TRYP对K562细胞作用机制研究:Rhdamine123荧光染色进行线粒体跨膜电位测定;利用SYBR Green RT-PCR方法检测TRYP作用K562细胞后凋亡相关基因Bcl-2、Bax、caspase-3 mRNA、融合基因bcr/abl mRNA的表达变化;Western blot法检测K562细胞凋亡相关蛋白Bcl-2、Bax、Cyt-c、caspase-3及融合蛋白bcr/abl的表达。
2.3 TRYP对小鼠K562白血病的作用及其机制研究:在体外抗肿瘤实验研究的基础上,以荷瘤小鼠为动物模型,观察TRYP对小鼠K562白血病的作用。采用CTX预处理,8×106个K562细胞/鼠尾静脉接种SCID小鼠建立白血病动物模型;50mg/kg、25mg/kgTRYP灌胃14d,观察动物的体质量,外周血白细胞数及其分类和骨髓涂片、bcr/abl mRNA表达、细胞周期变化、细胞表面抗原CD13检测,并取骨髓、心、肝、脾、肺、肾等重要脏器进行病理切片HE染色等,综合评价TRYP对白血病的治疗效果,并探讨其可能的作用机制。
3结果
3.1 TRYP对肿瘤细胞的抑制作用研究表明,在A549、HepG2、SW480、Eca-109、GRC-1、LNCAP、A375、K562等8种人源性肿瘤细胞中,K562细胞对TRYP的作用最为敏感,TRYP可明显抑制K562细胞增殖,且抑制作用呈现一定的浓度和时间依赖性。TRYP作用48h可诱导细胞形态和增殖周期发生明显改变;相差显微镜下,12.5μg/ml、25μg/ml TRYP组,细胞密度明显降低,大部分细胞边缘不清,亮度减弱,细胞出现溶解性死亡;透射电镜可见凋亡细胞增多,细胞核固缩,异染色质边集成新月状或环状,甚至坏死;Hoechst 33258染色荧光观察,正常组及溶媒对照组细胞核形态规则呈弥散均匀的荧光,而TRYP组细胞胞核内出现浓染致密的颗粒块状荧光,说明胞核固缩、染色质高度凝聚和碎裂。流式细胞术结果,TRYP可诱导K562细胞凋亡,48h后活细胞明显减少,凋亡和死亡细胞数明显上升,处于增殖活跃S期的细胞比例较对照组明显降低,尤其是25μg/ml TRYP组作用后,细胞S期比例降低至33.2%(P<0.01),G0/G1期、G2/M期细胞比例明显增高,分别上升至52.6%(P<0.05)、14.3%(P<0.01)。
3.2 TRYP抑制K562细胞的作用机制以6.25、12.5、25μg/ml TRYP处理K562细胞48 h后,细胞线粒体跨膜电位呈浓度依赖性下降,细胞中Bcl-2蛋白表达降低,而Bax蛋白的表达增强;胞浆中Cyt-c和细胞中caspase-3的表达也逐渐升高。其中以TRYP≥12.5μg/ml时,与空白对照相比作用显著;此外,25μg/ml TRYP还可抑制融合蛋白bcr/abl表达。利用SYBR Green RT-PCR方法检测上述蛋白在核酸水平的表达情况与Western blot方法检测到的结果较为一致。
3.3 TRYP对小鼠白血病的治疗作用采用CTX预注射和尾静脉接种K562细胞建立SCID小鼠白血病模型。接种K562细胞3w后,模型组动物体重明显减轻并整体呈现恶液质,外周血白细胞数升高,外周血及骨髓涂片可见中晚幼粒细胞。经50mg/kg、25mg/kgTRYP及180mg/kgHu(阳性对照药)治疗后,小鼠一般状况比模型组有所改善,体重下降程度轻于模型组。FCM检测给药组小鼠与模型组相比骨髓组织中CD13表达明显降低(P<0.01);骨髓中S期细胞比例下降;病理学观察发现给药后白血病细胞累及骨髓、脾、肝等脏器所引起的炎症反应有所改善,且50mg/kg组的疗效好于25mg/kg组。
4结论
采用荧光染色、透射电镜、流式细胞术等研究技术,证实TRYP对人白血病K562细胞及其诱发的白血病均具有显著抑制作用。其可能的作用机制,一是通过调节Bcl-2、Bax基因及蛋白的表达、降低线粒体跨膜电位、促使Cyt-c释放至胞浆,刺激下游分子caspase-3的活化引起K562细胞发生凋亡;二是TRYP通过抑制融合基因bcr/abl及蛋白的表达,导致K562细胞增殖抑制和凋亡。
Objective
Tryptanthrin, 6,12-dihydro-6,12-dioxoindolo-(2,1-b)-quinazoline, is a kind of indole quinazoline alkaloid. It is a yellow needle crystal and its melting point is between 267 and 268 centidegree. Tryptanthrin was isolated firstly from plants such as Strobilanthes cusia, Polygonum tinctorium Lour and Isatis tinctoria L. Literatures reported that tryptanthrin possessed anti-bacteria, anti-fungus, anti-inflammation, anti-tumor, anti-parasite activities and so on. But up to now, the researches of tryptanthrin mainly focused on the aspect of separation, extraction and synthesis because of the limited resource. The pharmacological activity studies of tryptanthrin mainly focused on anti-inflammation and little on anti-tumor. Morever, the study of tryptanthrin on hematological system tumor was only on acute promoyelocytic leukemia cells in vitro, there still has not the reports of proliferation, apoptosis and cell cycle effect of tryptanthrin on human chronic myeloid leukemia cell line-K562. In the study, tryptanthrin, synthesized by biomimetic synthesis method, was taken as study object to investigate its inhibitory activities and the action mechanism on human leukemia cell line-K562 adopting the neoplastic cells cultured in vitro and animal model bearing tumor in vivo, which provide the experimental data for the clinical application of tryptanthrin.
Methods
Part One: the anti-tumor effect of tryptanthrin on eight kinds of tumor cell and the study for tryptanthrin on inhibiting proliferation, inducing apoptosis of K562 in vitro By means of inhibiting proliferation assay, the anti-tumor sensitive activities of tryptanthrin were screened primarily on eight kinds of tumor cells including A549, HepG2, SW480, Eca-109, GRC-1, LNCAP, A375, K562. The tumor cell which is the most sensitive to tryptanthrin was ascerted as target cell. The concentration and time of tryptanthrin on the tumor cell were ascerted. The tumor cell in logarithmic growth phase was added tryptanthrin with different concentration to incubate for indicated time and then some indexes were detected to observe the change of morphology by contrast phase microscope, Hoechst 33258 fluorescent staining and transmission microscopy, analysis the cell apoptosis rate with Annexin-V-FITC and PI double labelings, and the change of cell cycle with PI simple staining by flow cytometer.
Part Two: the action mechanism of tryptanthrin on K562 cell in vitro The mitochondrial transmembrane potential was determined using Rhdamine 123 fluorescein stain by flow cytometer. After treated with tryptanthrin, the apoptosis relative gene expression of Bcl-2, Bax mRNA, caspase-3 and fusion gene bcr/abl mRNA in K562 cells were detected by SYBR Green Realtime PCR method. The expression of apoptosis relative protein Bcl-2, Bax, cytochrome c, caspase-3 and fusion protein bcr/abl were detected by Western blot.
Part Three: the treatment effect and action mechanism of tryptanthrin on leukemia-K562 mouse Combining with the anti-tumor effect data of tryptanthrin in vitro, the anti-tumor action mechanism of tryptanthrin in vivo was assessed using tumor-bearing mice SCID mouse. The general leukemia animal model was established adopting cytoxan pretreatment and vena caudalis injection with 8×106 K562 cells per mouse. After intragastric administration with 50mg/kg and 25mg/kg tryptanthrin for 14 days, the therapeutic effects of tryptanthrin were assessed by the change of body weight of animals, peripheral blood and bone marrow smear, leukocyte differential counting, the expression of bcr/abl mRNA, the analysis of cell cycle, the expression of cell surface antigen CD13, the expression of fusion gene bcr/abl mRNA. Meanwhile, the bone marrow, heart, liver, spleen, lung and kidney were taken to pathological section with haematoxylin and easin staining.
Results
First, the inhibitory effect of tryptanthrin on tumor cells MTT assay results confirmed that tryptanthrin was the most sensitive on K562 cell among eight kinds of tumor cells and could inhibit significantly on proliferation of K562 cells in a concentration and time manners. The cell morphous and proliferation cycle could be changed by tryptanthrin after 48 hours. At dose of 12.5μg/ml and 25μg/ml tryptanthrin, the cell density was decreased obviously and the morphologic change of K562 cell was altered significantly by contrast phase microscope, which exhibited ruptured cellular membrane, weaken brightness and even dissolved death of cells. Transmission electron microscopic results showed that the apoptosis cytosis, karyopyknosis, chromatin margination to crescent or circularity and cellular necrosis. Staining with Hoechst 33258 for fluorescence microscopy, some of the tryptanthrin-treated cells exhibited highly condensed and fragmented nuclei morphology, which were the typical characteristics of apoptosis. In contrast, the cells in the culture without tryptanthrin and dissolvent group respectively showed normal cell nuclei morphology with diffusion and uniform fluorescent light. The flow cytometer results indicated that tryptanthrin could not only induce apoptosis, but also influence the cell cycle. The normal cells decreased obviously, the proportion of apoptotic cells and necrotic cells increased significantly after treatment for 48 hours. With the concentration raised, viable apoptotic cell decreased and the proportion of non-viable apoptotic cell and necrotic cell increased obviously. The changes of cell cycle were obvious and the proportion of S phase decreased less than that of control group. Especially at the dose of 25μg/ml, the percent of S phase decreased to 33.2% (P<0.01). The cell population of the G0/G1 phase and G2/M phase was significantly increased up to 52.6% and 14.3% (P<0.05, P<0.01).
Second, the inhibitory effect mechanism of tryptanthrin on K562 cells Cells were treated with different concentrations of tryptanthrin and stained with Rhodamine 123 after 48 hours. Mitochondrial membrane potential of K562 cells decreased in a dose-dependent manner, which was consistent with the result of cell apoptosis. Western blot results indicated that the protein expression of Bcl-2 was downregulated and the expression levels of Bax were upregulated gradually after treatment with 6.25, 12.5 and 25μg/ml tryptanthrin. With the concentration raised, the expression of cytochrome c in cytoplasms and caspase-3 in cells were both increased. In addition, the expression of bcr/abl protein can be inhibited by tryptanthrin at the dose of 25μg/ml. The mRNA expression levels of Bcl-2, Bax, caspase3 and bcr/abl by SYBR Green PCR were in accordance with the protein expression levels by Western blot.
Third, the establishment of leukemia SCID mouse and the evaluation of therapeutic effect of tryptanthrin on leukemia SCID mouse The leukemia SCID animal model was established successfully adopting to cytoxan pretreatment and vena caudalis injection K562 cells, which exhibited the weight loss and wasting disease. The amount of peripheral white blood cell had a marked increasing after injection three weeks. Myelocyte and metagranulocyte could be observed in peripheral blood and bone marrow smear. After treatment with 25, 50 mg/kg tryptanthrin and 180 mg/kg hydroxyurea, the general state and the body weight of mouse was improved. The amount of peripheral white blood cell decreased. The FCM results showed that the expression of CD13 of bone marrow in tryptanthrin treatment group was decreased significantly compared with leukemia SCID group (P<0.01). The proportion of S phase of bone marrow cells was decreased. The pathology results indicated that the inflammation actions in organs induced by leukemia cells were improved to some extent. The therapeutic effect of 50 mg/kg tryptanthrin was better than that of 25 mg/kg tryptanthrin.
Conclusions
With fluorescein stain, transmission microscope and flow cytometer methods, the inhibitory effect of tryptanthrin was confirmed on K562 cell in vitro and leukemia induced by K562 cells in vivo. The action mechanism of tryptanthrin might on the one hand, regulate the expressions of Bcl-2, Bax mRNA and protein, degrade the mitochondrial transmembrane potential, make the cytochrome c release to cytoplasm and activate the downstream molecular caspase 3, on the other hand, inhibit the expression of bcr/abl mRNA and protein to suppress the proliferation and induce apoptosis of K562 cells.
色胺酮(Tryptanthrin,TRYP),为吲哚喹唑啉类生物碱,化学名称为吲哚[2,1-b]喹唑啉-6,12-二酮。黄色针晶,熔点267-268°C。最初由马蓝(Strobilanthes cusia)、蓼蓝(Polygonum tinctorium Lour)、菘蓝(Isatis tinctoria L.)等产蓝植物中得到。文献报道TRYP具有抗细菌真菌、抗炎、抗肿瘤及抗寄生虫等多种药理活性。但由于TRYP的来源有限,目前国外多集中在TRYP的分离、提取及合成,药理活性研究多集中在抗炎及少量抗肿瘤等方面。TRYP对血液系统肿瘤的作用也仅局限于对急性早幼粒白血病细胞的影响,但TRYP对慢性髓系白血病细胞增殖、凋亡、细胞周期等作用的研究尚未见报道。本课题以仿生合成得到的TRYP为研究试药,采用体外培养肿瘤细胞和建立体内动物模型等方法,探讨和揭示TRYP对人白血病细胞K562的体内外抑制作用及其机制研究,为TRYP的新药开发提供实验依据。
2方法
2.1 TRYP对8株人源性肿瘤细胞的MTT及抑制K562细胞增殖和诱导凋亡的研究:以A549、HepG2、SW480、Eca-109、GRC-1、LNCAP、A375、K562等8株人源性肿瘤细胞为靶细胞,以抑制增殖为指标,筛选TRYP对肿瘤细胞株的敏感性,然后以最敏感的肿瘤细胞为研究对象;同时,确定TRYP对该敏感细胞体外作用的药物浓度及作用时间;然后取对数生长期的细胞,分别以不同浓度TRYP作用一定时间后,收集细胞,分别采用相差显微镜、Hoechst 33258荧光染色法、透射电镜等方法观察细胞的形态学改变,Annexin-V- FITC和PI双标记流式细胞术检测细胞凋亡率;PI单染色检测细胞周期的变化。
2.2 TRYP对K562细胞作用机制研究:Rhdamine123荧光染色进行线粒体跨膜电位测定;利用SYBR Green RT-PCR方法检测TRYP作用K562细胞后凋亡相关基因Bcl-2、Bax、caspase-3 mRNA、融合基因bcr/abl mRNA的表达变化;Western blot法检测K562细胞凋亡相关蛋白Bcl-2、Bax、Cyt-c、caspase-3及融合蛋白bcr/abl的表达。
2.3 TRYP对小鼠K562白血病的作用及其机制研究:在体外抗肿瘤实验研究的基础上,以荷瘤小鼠为动物模型,观察TRYP对小鼠K562白血病的作用。采用CTX预处理,8×106个K562细胞/鼠尾静脉接种SCID小鼠建立白血病动物模型;50mg/kg、25mg/kgTRYP灌胃14d,观察动物的体质量,外周血白细胞数及其分类和骨髓涂片、bcr/abl mRNA表达、细胞周期变化、细胞表面抗原CD13检测,并取骨髓、心、肝、脾、肺、肾等重要脏器进行病理切片HE染色等,综合评价TRYP对白血病的治疗效果,并探讨其可能的作用机制。
3结果
3.1 TRYP对肿瘤细胞的抑制作用研究表明,在A549、HepG2、SW480、Eca-109、GRC-1、LNCAP、A375、K562等8种人源性肿瘤细胞中,K562细胞对TRYP的作用最为敏感,TRYP可明显抑制K562细胞增殖,且抑制作用呈现一定的浓度和时间依赖性。TRYP作用48h可诱导细胞形态和增殖周期发生明显改变;相差显微镜下,12.5μg/ml、25μg/ml TRYP组,细胞密度明显降低,大部分细胞边缘不清,亮度减弱,细胞出现溶解性死亡;透射电镜可见凋亡细胞增多,细胞核固缩,异染色质边集成新月状或环状,甚至坏死;Hoechst 33258染色荧光观察,正常组及溶媒对照组细胞核形态规则呈弥散均匀的荧光,而TRYP组细胞胞核内出现浓染致密的颗粒块状荧光,说明胞核固缩、染色质高度凝聚和碎裂。流式细胞术结果,TRYP可诱导K562细胞凋亡,48h后活细胞明显减少,凋亡和死亡细胞数明显上升,处于增殖活跃S期的细胞比例较对照组明显降低,尤其是25μg/ml TRYP组作用后,细胞S期比例降低至33.2%(P<0.01),G0/G1期、G2/M期细胞比例明显增高,分别上升至52.6%(P<0.05)、14.3%(P<0.01)。
3.2 TRYP抑制K562细胞的作用机制以6.25、12.5、25μg/ml TRYP处理K562细胞48 h后,细胞线粒体跨膜电位呈浓度依赖性下降,细胞中Bcl-2蛋白表达降低,而Bax蛋白的表达增强;胞浆中Cyt-c和细胞中caspase-3的表达也逐渐升高。其中以TRYP≥12.5μg/ml时,与空白对照相比作用显著;此外,25μg/ml TRYP还可抑制融合蛋白bcr/abl表达。利用SYBR Green RT-PCR方法检测上述蛋白在核酸水平的表达情况与Western blot方法检测到的结果较为一致。
3.3 TRYP对小鼠白血病的治疗作用采用CTX预注射和尾静脉接种K562细胞建立SCID小鼠白血病模型。接种K562细胞3w后,模型组动物体重明显减轻并整体呈现恶液质,外周血白细胞数升高,外周血及骨髓涂片可见中晚幼粒细胞。经50mg/kg、25mg/kgTRYP及180mg/kgHu(阳性对照药)治疗后,小鼠一般状况比模型组有所改善,体重下降程度轻于模型组。FCM检测给药组小鼠与模型组相比骨髓组织中CD13表达明显降低(P<0.01);骨髓中S期细胞比例下降;病理学观察发现给药后白血病细胞累及骨髓、脾、肝等脏器所引起的炎症反应有所改善,且50mg/kg组的疗效好于25mg/kg组。
4结论
采用荧光染色、透射电镜、流式细胞术等研究技术,证实TRYP对人白血病K562细胞及其诱发的白血病均具有显著抑制作用。其可能的作用机制,一是通过调节Bcl-2、Bax基因及蛋白的表达、降低线粒体跨膜电位、促使Cyt-c释放至胞浆,刺激下游分子caspase-3的活化引起K562细胞发生凋亡;二是TRYP通过抑制融合基因bcr/abl及蛋白的表达,导致K562细胞增殖抑制和凋亡。
Objective
Tryptanthrin, 6,12-dihydro-6,12-dioxoindolo-(2,1-b)-quinazoline, is a kind of indole quinazoline alkaloid. It is a yellow needle crystal and its melting point is between 267 and 268 centidegree. Tryptanthrin was isolated firstly from plants such as Strobilanthes cusia, Polygonum tinctorium Lour and Isatis tinctoria L. Literatures reported that tryptanthrin possessed anti-bacteria, anti-fungus, anti-inflammation, anti-tumor, anti-parasite activities and so on. But up to now, the researches of tryptanthrin mainly focused on the aspect of separation, extraction and synthesis because of the limited resource. The pharmacological activity studies of tryptanthrin mainly focused on anti-inflammation and little on anti-tumor. Morever, the study of tryptanthrin on hematological system tumor was only on acute promoyelocytic leukemia cells in vitro, there still has not the reports of proliferation, apoptosis and cell cycle effect of tryptanthrin on human chronic myeloid leukemia cell line-K562. In the study, tryptanthrin, synthesized by biomimetic synthesis method, was taken as study object to investigate its inhibitory activities and the action mechanism on human leukemia cell line-K562 adopting the neoplastic cells cultured in vitro and animal model bearing tumor in vivo, which provide the experimental data for the clinical application of tryptanthrin.
Methods
Part One: the anti-tumor effect of tryptanthrin on eight kinds of tumor cell and the study for tryptanthrin on inhibiting proliferation, inducing apoptosis of K562 in vitro By means of inhibiting proliferation assay, the anti-tumor sensitive activities of tryptanthrin were screened primarily on eight kinds of tumor cells including A549, HepG2, SW480, Eca-109, GRC-1, LNCAP, A375, K562. The tumor cell which is the most sensitive to tryptanthrin was ascerted as target cell. The concentration and time of tryptanthrin on the tumor cell were ascerted. The tumor cell in logarithmic growth phase was added tryptanthrin with different concentration to incubate for indicated time and then some indexes were detected to observe the change of morphology by contrast phase microscope, Hoechst 33258 fluorescent staining and transmission microscopy, analysis the cell apoptosis rate with Annexin-V-FITC and PI double labelings, and the change of cell cycle with PI simple staining by flow cytometer.
Part Two: the action mechanism of tryptanthrin on K562 cell in vitro The mitochondrial transmembrane potential was determined using Rhdamine 123 fluorescein stain by flow cytometer. After treated with tryptanthrin, the apoptosis relative gene expression of Bcl-2, Bax mRNA, caspase-3 and fusion gene bcr/abl mRNA in K562 cells were detected by SYBR Green Realtime PCR method. The expression of apoptosis relative protein Bcl-2, Bax, cytochrome c, caspase-3 and fusion protein bcr/abl were detected by Western blot.
Part Three: the treatment effect and action mechanism of tryptanthrin on leukemia-K562 mouse Combining with the anti-tumor effect data of tryptanthrin in vitro, the anti-tumor action mechanism of tryptanthrin in vivo was assessed using tumor-bearing mice SCID mouse. The general leukemia animal model was established adopting cytoxan pretreatment and vena caudalis injection with 8×106 K562 cells per mouse. After intragastric administration with 50mg/kg and 25mg/kg tryptanthrin for 14 days, the therapeutic effects of tryptanthrin were assessed by the change of body weight of animals, peripheral blood and bone marrow smear, leukocyte differential counting, the expression of bcr/abl mRNA, the analysis of cell cycle, the expression of cell surface antigen CD13, the expression of fusion gene bcr/abl mRNA. Meanwhile, the bone marrow, heart, liver, spleen, lung and kidney were taken to pathological section with haematoxylin and easin staining.
Results
First, the inhibitory effect of tryptanthrin on tumor cells MTT assay results confirmed that tryptanthrin was the most sensitive on K562 cell among eight kinds of tumor cells and could inhibit significantly on proliferation of K562 cells in a concentration and time manners. The cell morphous and proliferation cycle could be changed by tryptanthrin after 48 hours. At dose of 12.5μg/ml and 25μg/ml tryptanthrin, the cell density was decreased obviously and the morphologic change of K562 cell was altered significantly by contrast phase microscope, which exhibited ruptured cellular membrane, weaken brightness and even dissolved death of cells. Transmission electron microscopic results showed that the apoptosis cytosis, karyopyknosis, chromatin margination to crescent or circularity and cellular necrosis. Staining with Hoechst 33258 for fluorescence microscopy, some of the tryptanthrin-treated cells exhibited highly condensed and fragmented nuclei morphology, which were the typical characteristics of apoptosis. In contrast, the cells in the culture without tryptanthrin and dissolvent group respectively showed normal cell nuclei morphology with diffusion and uniform fluorescent light. The flow cytometer results indicated that tryptanthrin could not only induce apoptosis, but also influence the cell cycle. The normal cells decreased obviously, the proportion of apoptotic cells and necrotic cells increased significantly after treatment for 48 hours. With the concentration raised, viable apoptotic cell decreased and the proportion of non-viable apoptotic cell and necrotic cell increased obviously. The changes of cell cycle were obvious and the proportion of S phase decreased less than that of control group. Especially at the dose of 25μg/ml, the percent of S phase decreased to 33.2% (P<0.01). The cell population of the G0/G1 phase and G2/M phase was significantly increased up to 52.6% and 14.3% (P<0.05, P<0.01).
Second, the inhibitory effect mechanism of tryptanthrin on K562 cells Cells were treated with different concentrations of tryptanthrin and stained with Rhodamine 123 after 48 hours. Mitochondrial membrane potential of K562 cells decreased in a dose-dependent manner, which was consistent with the result of cell apoptosis. Western blot results indicated that the protein expression of Bcl-2 was downregulated and the expression levels of Bax were upregulated gradually after treatment with 6.25, 12.5 and 25μg/ml tryptanthrin. With the concentration raised, the expression of cytochrome c in cytoplasms and caspase-3 in cells were both increased. In addition, the expression of bcr/abl protein can be inhibited by tryptanthrin at the dose of 25μg/ml. The mRNA expression levels of Bcl-2, Bax, caspase3 and bcr/abl by SYBR Green PCR were in accordance with the protein expression levels by Western blot.
Third, the establishment of leukemia SCID mouse and the evaluation of therapeutic effect of tryptanthrin on leukemia SCID mouse The leukemia SCID animal model was established successfully adopting to cytoxan pretreatment and vena caudalis injection K562 cells, which exhibited the weight loss and wasting disease. The amount of peripheral white blood cell had a marked increasing after injection three weeks. Myelocyte and metagranulocyte could be observed in peripheral blood and bone marrow smear. After treatment with 25, 50 mg/kg tryptanthrin and 180 mg/kg hydroxyurea, the general state and the body weight of mouse was improved. The amount of peripheral white blood cell decreased. The FCM results showed that the expression of CD13 of bone marrow in tryptanthrin treatment group was decreased significantly compared with leukemia SCID group (P<0.01). The proportion of S phase of bone marrow cells was decreased. The pathology results indicated that the inflammation actions in organs induced by leukemia cells were improved to some extent. The therapeutic effect of 50 mg/kg tryptanthrin was better than that of 25 mg/kg tryptanthrin.
Conclusions
With fluorescein stain, transmission microscope and flow cytometer methods, the inhibitory effect of tryptanthrin was confirmed on K562 cell in vitro and leukemia induced by K562 cells in vivo. The action mechanism of tryptanthrin might on the one hand, regulate the expressions of Bcl-2, Bax mRNA and protein, degrade the mitochondrial transmembrane potential, make the cytochrome c release to cytoplasm and activate the downstream molecular caspase 3, on the other hand, inhibit the expression of bcr/abl mRNA and protein to suppress the proliferation and induce apoptosis of K562 cells.
引文
[1] Schindler F, Zahner H. Metabolic products of microorganisms.91. Tryp- tanthrin, a tryptophan derived antibiotic from Candida lipolytica. Arch Mikrobiol. 1971, 79(3):187-203
[2] Danz H, Baumann D, Hamburger M. Quantitative determination of the dual COX-2/5-LOX inhibitor tryptanthrin in Isatis tinctoria by ESI-LC- MS. Planta Med. 2002, 68(2):152-7
[3] Honda G, Tabata M. Isolation of Antifungal Principle Tryptanthrin from Strobilanthes cusia O. Kuntze [J]. Planta Medica, 1979, 36:85-6
[4] Honda G, Tosirisuk V, Tabata M. Isolation of an antidermatophytic, tryptanthrin, from indigo plants, Polygonum tinctorium and Isatis tinctoria [J]. Planta Medica, 1980, 38(3):275-6
[5] Mohn T, Potterat O, Hamburger M. Quantification of active principles and pigments in leaf extracts of Isatis tinctoria by HPLC/UV/MS. Planta Med. 2007, 73(2):151-6
[6] Yoshikawa M, Murakami T, Akinobu K, et al. Novel indole S,O-bisdes- moside, calanthoside, the precursor glycoside of tryptanthrin, indirubin, and isatin, with increasing skin blood flow promoting effects, from two Calanthe species (Orchidaceae)[J]. Chem Plarm Bull(Tokyo), 1998, 46(5): 886-8
[7]阮金兰,邹建华,蔡亚玲.大青叶化学成分研究.中国中药杂志,2005,30(19):1525-6
[8]李彬,陈万生,郑水庆等.四倍体板蓝根中的两个新生物碱.药学学报,2000, 35(7):508-10
[9] Oberthür C, Hamburger M. Tryptanthrin content in Isatis tinctoria leaves--a comparative study of selected strains and post-harvest treatments. Planta Med.2004,70(7):642-5
[10] Liau BC, Jong TT, Lee MR. LC-APCI-MS method for detection and analysis of tryptanthrin, indigo, and indirubin in daqingye and banlangen [J]. Pharm Biomed Anal. 2007,43(1):346-51
[11] Hosoe T, Nozawa K, Kawahara N, et al. Isolation of a new potent cytotoxic pigment along with indigotin from the pathogenic basidiomycetous fungus Schizophyllum commune [J]. Mycopathologia, 1999,144(1):9-12
[12]李清华,金继曙,种明才等.青黛抗真菌成份的研究.中草药,1983,14(10):8-9
[13] Eung Seok Lee, Jae-Gyu Park, Yurngdong Jahng. A facile synthesis of simple alkaloids-synthesis of 2,3-polymethylene-4(3H)-quinazolinones and related alkaloids. Tetrahedron Letters, 2003,(44):1883-6
[14] Witt A, Bergman J. Recent development in the field of quinazoline chemistry [J]. Curreng Ory. Chem, 2003,7:659-77
[15]王翠玲,刘建利,沈小莉.天然产物色胺酮及其衍生物合成的研究进展.化学通报. 2007,70(2):89-95
[16] Kataoka M, Hirata K, Kunikata T, et al. Antibacterial action of tryptanthrin and kaempferol, isolated from the indigo plant (Polygonum tinctorium Lour.), against Helicobacter pylori-infected Mongolian gerbils. [J] Gastroenterol, 2001, 36(1):5-9
[17] Oberthür C, J?ggi R, Hamburger M. HPLC based activity profiling for 5-lipoxygenase inhibitory activity in Isatis tinctoria leaf extracts. Fitoterapia. 2005, 76(3-4):324-32
[18] Tatsuya I, Keizo K, Shimpei U, et al. Tryptanthrin inhibits nitric oxide and prostaglandin E2 synthesis by murine macrophages [J].European Journal of Pharmacology, 2000, 407:197-204
[19] Mark J M, Kanso I, Tatsuya I, et al. The natural plant product tryptanthrin ameliorates dextran sodium sulfate-induced colitis in mice. InternationalImmunopharmacology, 2002, (2):565-78
[20] Takei Y, Kunikata T, Aga M, et al. Tryptanthrin inhibits interferon-gamma production by Peyer's patch lymphocytes derived from mice that had been orally administered staphylococcal enterotoxin. Biol Pharm Bull. 2003,26(3): 365-7
[21] Danz H, Stoyanova S, Wippich P, et al. Identification and isolation of the cyclooxygenase-2 inhibitory principle in Isatis tinctoria [J]. Planta Med. 2001, 67 (5):411-6
[22] Danz H, Stoyanova S, Thomet OA, et al. Inhibitory activity of tryptanthrin on prostaglandin and leukotriene synthesis[J]. Planta Med. 2002,68(10):875-80
[23]王振义.白血病的诱导凋亡疗法[N].中国医学论坛报,2003,29(1):72-5
[24] Kimoto T, Hino K, Koya-Miyata S, et al. Cell differentiation and apoptosis of monocytic and promyelocytic leukemia cells (U-937 and HL-60) by tryptanthrin, an active ingredient of Polygonum tinctorium Lour [J]. Pathology international, 2001, 51(5):315-25
[25] Mu Ben-zhefu. A new cytotoxic active principle isolated from Polygonum tinctorium. Natural Medicine, 1999, 53(2):72-9
[26] Motoki T, Takami Y, Yagi Y, et al. Inhibition of hepatocyte growth factor induction in human dermal fibroblasts by tryptanthrin. Biol Pharm Bull, 2005, 28(2):260-6
[27] Sharma VM, Prasanna P, Seshu KV, et al. Novel indolo [2,1-b]quinazoline analogues as cytostatic agents: synthesis, biological evaluation and structure- activity relationship. Bioorg Med Chem Lett. 2002,12(17):2303-7
[28] Sung-Tsai Yu, Tzer-Ming Chen, Shih-Yun Tseng, et al. Tryptanthrin inhibits MDR1 and reverses doxorubicin resistance in breast cancer cells. Biochemical and Biophysical Research Communications. 2007, 358:79-84
[29] Koya-Miyata S, Kimoto T, Micallef MJ, et al. Prevention of azoxymethane- induced intestinal tumors by a crude ethyl acetate-extract and tryptanthrin extracted from Polygonum tinctorium Lour [J]. Anti-cancer Research, 2001,21(5):3295-300
[30] Bhattacharjee AK, Hartell MG, Nichols DA, et al. Structure-activity relationship study of antimalarial indolo [2,1-b]quinazoline-6,12-diones (tryptanthrins). Three dimensional harmacophore modeling and identification of new antimalarial candidates. Eur J Med Chem. 2004,39(1):59-67
[31] Bhattacharjee AK, Skanchy DJ, Jennings B, et al. Analysis of stereo- electronic properties, mechanism of action and pharmacophore of synthetic indolo[2,1-b] quinazoline-6,12-dione derivatives in relation to antileishmanial activity using quantum chemical, cyclic voltammetry and 3-D-Qsar catalyst procedures. J. Bioorg Med Chem. 2002,10: 1979-89
[32] Scovill J, Blank E, Konnick M, et al. Antitrypanosomal activities of tryptanthrins. Antimicrob Agents Chemother. 2002, 46(3):882-3
[33] Green DR, Reed JC. Mitochondria and apoptosis [J]. Science, 1998, 281(5381): 1309-12.
[34] Glaser T, Weller M. Caspase-dependent chemotherapy induced death of glioma cells requires mitochondrial cytochrome c release [J]. Biophys Res Commun, 2001, 281 (2): 322-7.
[35] Newmeyer D, Ferguson MS. Mitochondria: releasing power for life and unleashing the machineries of death [J]. Cell, 2003, 112 (4): 481-90.
[36] Armstrong JS. Mitochondrial membrane permeabilization: the sine quanon for cell death [J]. Bioessays, 2006, 28 (3): 253-60.
[37] Zamzami N, Kroemer G. The mitochondrion in apoptosis: how Pandora’s box opens [J]. Nat Rev Mol Cell Biol, 2001, 2 (1): 67-71.
[38] Saris NE, Carafoli E. A historical review of cellular calcium handling with emphasis on mitochondria [J]. Biochemistry (Moscow), 2005, 70(2): 187-94.
[39] Linda E, Frank AE, Kruyt E, et al. Cell death independent of caspases: A review [J]. Clin Cancer Res, 2005, 11(9): 3155-62.
[40] Scorrano L, Ashiya M, Buttle K, et al. A distinct pathway remodelsmitochondrial cristae and mobilizes cytochrome c during apoptosis [J]. Dev Cell, 2002, 2 (1): 55-67.
[41] Loo G, Saelens X, Gurp M, et al. The role of mitochondrial factors in apoptosis: a russian roulette with more than one bullet [J]. Cell Death Differ, 2002, 9 (10):1031-42.
[42] Hatai T, Matsuzawa A, Inoshita S, et al. Execution of apoptosis signal- regulating kinase1 (ASK1) induced apoptosis by the mitochondria- dependent caspase activation [J]. J Biol Chem, 2000, 275 (34): 26576-81.
[43] Green D R. Apoptotic pathways: the roads to ruin [J]. Cell, 1998, 94 (6): 695-8.
[44] Chang H Y, Yang X. Proteases for cell suicide: functions and regulation of caspases [J]. Microbiol Mol Biol Rev, 2000, 64 (4): 821-46
[45] Hsu SY, Hsueh AJ. Tissue-specific Bcl-2 protein partners in apoptosis: An ovarian paradigm [J]. Physiol Rev, 2000, 80 (2): 593-614.
[46] Schindler CK, Shinoda S, Simon RP, et al. Subcellular distribution of Bcl-2 family proteins and 14-3-3 within the hippocampus during seizure-induced neuronal death in the rat [J]. Neurosci Lett, 2004, 356(3): 163-6.
[47] Gross A, McDonnell JM, Korsmeyer SJ. Bcl-2 family members and the mitochondria in apoptosis [J]. Genes Dev, 1999, 13(15): 1899-911.
[48] Burlacu A. Regulation of apoptosis by Bcl-2 family proteins [J]. J Cell Mol Med, 2003, 7 (3): 249-57.
[49] Puthalakath H, Strasser A. Keeping killers on a tight leash: transcriptional and post-translational control of the pro-apoptotic activity of BH3-only proteins [J]. Cell Death Differ, 2002, 9 (5): 505-12.
[50] Tsujimoto Y. Cell death regulation by the Bcl-2 protein family in the mitoehondria [J]. J Cell Physiol, 2003, 195 (2): 158-67.
[51] Yin XM, Oltvai ZN, Korsmeyer SJ. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax [J]. Nature, 1994, 369 (6478): 321-3.
[52] Sattler M, Liang H, Nettesheim D, et al. Structure of Bcl-xL-Bak peptide complex: recognition between regulators of apoptosis [J]. Science, 1997, 275 (5302): 983-6.
[53] Petros AM, Nettesheim DG, Wang Y, et al. Rationale for Bcl-xL/Bad peptide complex formation from structure, mutagenesis and biophysical studies [J]. Protein Sci, 2000, 9(12): 2528-34.
[54] Orrenius S. Mitochondrial regulation of apoptotic cell death [J]. Toxicol Lett, 2004, 149 (123): 19-23.
[55] Chou JJ, Li H, Salvesen GS, et al. Solution structure of BID, an intracellular amplifier of apoptotic signaling [J]. Cell, 1999,96 (5): 615-24.
[56] Kuwana T, Mackey MR, Perkins G, et al. Bid, Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane [J]. Cell, 2002, 111(3): 331-42.
[57] Saito M, Korsmeyer SJ, Schlesinger PH. Bax-dependent transport of cytochrome c reconstituted in pure liposomes [J]. Nat Cell Biol, 2000, 2(8): 553-5.
[58] Haeberlein SL.Mitochondrial functions in apoptotic neuronal cell death [J]. Neurochem Res, 2004, 29(3): 521-30.
[59] Wu C, Fujihara H, Yao J, et al. Different expression patterns of Bcl-2, Bcl-xl, and Bax proteins after sublethal forebrain ischemia in C57Black/Crj6 mouse striatum [J]. Stroke, 2003, 34(7): 1803-8.
[60] Susin SA, Lorenzo HK, ZamzamiN, et al. Molecular characterization of mitochondrial apoptosis inducing factor [J]. Nature, 1999, 39 (7): 441-6.
[61] Skulachev VP. Cytochrome c in the apoptotic and antioxidant cascades [J]. FEBS Lett, 1998, 423 (3): 275-80.
[62] Vieira H, Kroemer G. Mitochondria as targets of apoptosis regulation by nitric oxide [J]. IUBMB Life, 2003, 55(10211): 613-20.
[63] Li K, Li Y, Shelton JM, et al. Cytochrome c deficiency causes embryonic lethality and attenuates stress-induced apoptosis [J]. Cell, 2000, 101(4): 389-9.
[64] Bras M,Queenan B, Susin SA. Programmed cell death via mitochondria:different modes of dying [J]. Biochemistry (Moscow), 2005, 70(2): 231-9.
[65] Rice AM, Wood JA, Milrose CG, et al. Conditions that enable human hematopoietic stem cell engraftment in all NOD-SCID mice [J]. Transplantation, 2000, 69 (5): 927-35.
[66] Chargui J, Moya MJ, Sanhadji K, et al. Anti-NK antibodies injected into recipient mice enhance engraftment and chimerism after allogeneic transplantation of fetal liver stem cells [J]. Thymus, 1997, 24 (4):233-46.
[67] Kollet O, Peled A, Byk T, et al . B-Microglobulin-deficient (B-mnull) NOD/SCID mice are excellent recipients for studying human stem cell function [J]. Blood, 2000, 95(10):3102-5.
[68] Ohsugi T, Yamaguchi K, Kumasaka T, et al. Rapid tumor death model for evaluation of new therapeutic agents for adult T-cell leukemia [J]. Lab Invest, 2004,84(2):263-6.
[69] Feuring-Buske M, Gerhard B, Cashman J, et al. Improved engraftment of human acute myeloid leukemia progenitor cells in beta 2-micro- globulin-deficient NOD/SCID mice transgenic for human growth factors[J].Leukemia, 2003,17(4):760-3.
[70] Mitchell P, Lee FT, Hall C, et al. Targeting primary human Ph(+) B-cell precursor leukemia engrafted SCID mice using radiolabeled anti-CD19 monoclonal antibodies[J]. J Nucl Med, 2003, 44 (7):1105-16.
[71] Dazzi F, Hasserjian R, Gordon MY, et al. Normal and chronic phase CML hematopoietic cells repopulate NOD/SCID bone marrow with different kinetics and cell lineage representation[J]. Hematol J, 2000,1(5):307-15.
[72] Peled A, Hardan I, Trakhtenbrot L, et al. Immature leukemic CD34+CX- CR4+ cells from CML patients have lower integrin-dependent migration and adhesion in response to the chemokine SDF-1[J]. Stem Cells, 2002, 20(3):259-66.
[73] Ramirez M, Diaz MA, Madero L, et al. Transplantation of marrow cells from children with standard risk-acute lymphoblastic leukemia at the endof therapy into NOD/SCID mice for detecting residual leukemic cells with in vivo growth potential [J]. Leuk Res, 2003, 27(12): 1153-7.
[74] Herrera L, Yarbrough S, Ghetie V, et al. Treatment of SCID/human Bcell precursor ALL with anti-CD19 and anti-CD22 immunotoxins [J]. Leukemia, 2003, 17(2):334-338.
[75] Golay J, Di Gaetano N, Amico D, et al. Gemtuzumab ozogamicin (Mylotarg) has therapeutic activity against CD33 acute lymphoblastic leukaemias in vitro and in vivo [J]. Br J Haematol, 2005, 128(3):310-7.
[76] Nijmeijer BA, Willemze R. An animal model for human cellular immunotherapy: specific eradication of human acute lymphoblastic leukemia by cytotoxic T lymphocytes in NOD/SCID mice [J]. Blood, 2002, 100 (2):654-7.
[77] Honma Y, Ishii Y, Sassa T, et al. Treatment of human promyelocytic leukemia in the SCID mouse model with cotylenin A, an inducer of myelomonocytic differentiation of leukemia cells[J]. Leuk Res, 2003, 27(11):1019-25.
[78] Levis M, Murphy KM, Pham R, et al. Internal tandem duplications of the FLT3 gene are present in leukemia stem cells [J]. Blood, 2005, 106(2): 673-80.
[79] Piloto O, Levis M, Huso D, et al. Inhibitory anti-FLT3 antibodies are capable of mediating antibody-dependent cell-mediated cytotoxicity and reducing engraftment of acute myelogenous leukemia blasts in nonobese diabetic/severe combined immunodeficient mice [J]. Cancer Res, 2005, 65(4):1514-35.
[80] Bai L, Kon K, Tatsumi M, et al. A human B-cell model established by transplantation of JOK-1 cells into SCID mice and an anti-leukemia efficacy of fludarabine phosphate [J]. Oncol Rep, 2000, 7(1):33-8.
[81] Zhang Y, Dawson MI, Mohammsd R, et al. Indeuction of apoptosis of human B-CLL and ALL cells by a novel retinoid and its nonretinoidalanalog [J].Blood, 2002, 100(8):2917-25.
[82] Garattini E, Parrella E, Diomede L, et al. ST1926, a novel and orally active retinoid-related molecule inducing apoptosis in myeloid leukemia cells: modulation of intracellular calcium homeostasis [J]. Blood, 2004, 103(1):194-207.
[83] Kerr JFR, Wyllie, Currie AR. Apoptosis, a basic biologic phenomenon with wide implication in tissue kinetics.Brit. J.Cancer. 1972, 26: 239-45
[84] YANG J, LIU X, BHALLA K, etal. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked [J]. Seienee, 1997, 275(5303): 1129-32
[85] Gorczyca W, Gong J, Ardelt B, et al. The cell cycle related differences in susceptilility of HL-60 cells to apoptosis induced by various antitumor agents. Cancer Res. 1993,53:3186-92
[86] Sun MG, Williams J, Munoz-Pinedo C, Perkins GA, Brown JM, Ellisman MH, Green DR, Frey TG. Correlated three-dimensional light and electron microscopy reveals transformation of mitochondria during apoptosis. Nat Cell Biol 2007, 9:1057-65
[87] Sareen D, Darjatmoko SR, Albert DM, Polans AS. Mitochondria, calcium, and calpain are key mediators of resveratrol-induced apoptosis in breast cancer. Mol Pharmacol 2007, 72: 1466-75
[88] Hortelano, S. and Bosca, L. 6-Mercptopurine decreases the Bcl-2/Bax ratio and induces apoptosis in activated splenic B lymphocytes. Mol. Pharmcol. 1997, 51, 414-21.
[89] Oltvai, Z.N., Milliman, C.L. and Korsmeyer, S.J. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell, 1993, 74: 609-19.
[90] Green DR, Reed JC.Mitochondria and apoptosis. Science.1998, 281(5381): 1309
[91] Brener C, Cadiou H, Vieira HL, et al.Bcl-2 and Bax regulate the channelactivity of the mitochondrial adenine nucleotide translocator. Oncogene, 2000, 19(3): 329-36
[92] Shimizu S, Narita M, Tsujimoto Y. Bcl-2 family proteins regulate the release of apopotogenic cytochrome c by the mitochondrial channel VDAC. Nature, 1999, 399(6735): 483-7
[93] Shimiz S, Ide T, Yanagida T, et al. Electrophysiological study of a novel large pore formed by Bax and the voltage-dependent anion channel that is permeable to cytochrome c.[J] Biol Chem, 2000, 275(16): 12321-5
[94] Li P, Nijhawan D, Budihardjo I, et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell,1997, 91(4):479-89
[95]缪珊,张海,石小鹏,孙纪元,周宣暄,谢艳华,王捷频,苗青,王四旺.色胺酮对人白血病细胞株K562细胞增殖抑制、凋亡诱导的影响.中国药理学通报. 2009,25(2):256-8
[96]陈奇主编.中药药理实验方法北京人民卫生出版社1994:206
[97]沈志祥,陈钰,李军民编著.简明临床血液病学.上海:上海科学技术文献出版社. 2002. 6:8,506
[98] Walter DH, Haendeler J, Galle J, et al. Cyclosporin A inhibits apoptosis of human endothelialcells by preventing release of cytochrome c from mitochondria [J]. Circulation, 1998, 98: 1153
[99] Leckie BJ. Targeting the renin-angiotensin system: what’s new. [J] Curr Med Chem Cardiovasc Hematol Agents, 2005, 3(1): 23-32.
[2] Danz H, Baumann D, Hamburger M. Quantitative determination of the dual COX-2/5-LOX inhibitor tryptanthrin in Isatis tinctoria by ESI-LC- MS. Planta Med. 2002, 68(2):152-7
[3] Honda G, Tabata M. Isolation of Antifungal Principle Tryptanthrin from Strobilanthes cusia O. Kuntze [J]. Planta Medica, 1979, 36:85-6
[4] Honda G, Tosirisuk V, Tabata M. Isolation of an antidermatophytic, tryptanthrin, from indigo plants, Polygonum tinctorium and Isatis tinctoria [J]. Planta Medica, 1980, 38(3):275-6
[5] Mohn T, Potterat O, Hamburger M. Quantification of active principles and pigments in leaf extracts of Isatis tinctoria by HPLC/UV/MS. Planta Med. 2007, 73(2):151-6
[6] Yoshikawa M, Murakami T, Akinobu K, et al. Novel indole S,O-bisdes- moside, calanthoside, the precursor glycoside of tryptanthrin, indirubin, and isatin, with increasing skin blood flow promoting effects, from two Calanthe species (Orchidaceae)[J]. Chem Plarm Bull(Tokyo), 1998, 46(5): 886-8
[7]阮金兰,邹建华,蔡亚玲.大青叶化学成分研究.中国中药杂志,2005,30(19):1525-6
[8]李彬,陈万生,郑水庆等.四倍体板蓝根中的两个新生物碱.药学学报,2000, 35(7):508-10
[9] Oberthür C, Hamburger M. Tryptanthrin content in Isatis tinctoria leaves--a comparative study of selected strains and post-harvest treatments. Planta Med.2004,70(7):642-5
[10] Liau BC, Jong TT, Lee MR. LC-APCI-MS method for detection and analysis of tryptanthrin, indigo, and indirubin in daqingye and banlangen [J]. Pharm Biomed Anal. 2007,43(1):346-51
[11] Hosoe T, Nozawa K, Kawahara N, et al. Isolation of a new potent cytotoxic pigment along with indigotin from the pathogenic basidiomycetous fungus Schizophyllum commune [J]. Mycopathologia, 1999,144(1):9-12
[12]李清华,金继曙,种明才等.青黛抗真菌成份的研究.中草药,1983,14(10):8-9
[13] Eung Seok Lee, Jae-Gyu Park, Yurngdong Jahng. A facile synthesis of simple alkaloids-synthesis of 2,3-polymethylene-4(3H)-quinazolinones and related alkaloids. Tetrahedron Letters, 2003,(44):1883-6
[14] Witt A, Bergman J. Recent development in the field of quinazoline chemistry [J]. Curreng Ory. Chem, 2003,7:659-77
[15]王翠玲,刘建利,沈小莉.天然产物色胺酮及其衍生物合成的研究进展.化学通报. 2007,70(2):89-95
[16] Kataoka M, Hirata K, Kunikata T, et al. Antibacterial action of tryptanthrin and kaempferol, isolated from the indigo plant (Polygonum tinctorium Lour.), against Helicobacter pylori-infected Mongolian gerbils. [J] Gastroenterol, 2001, 36(1):5-9
[17] Oberthür C, J?ggi R, Hamburger M. HPLC based activity profiling for 5-lipoxygenase inhibitory activity in Isatis tinctoria leaf extracts. Fitoterapia. 2005, 76(3-4):324-32
[18] Tatsuya I, Keizo K, Shimpei U, et al. Tryptanthrin inhibits nitric oxide and prostaglandin E2 synthesis by murine macrophages [J].European Journal of Pharmacology, 2000, 407:197-204
[19] Mark J M, Kanso I, Tatsuya I, et al. The natural plant product tryptanthrin ameliorates dextran sodium sulfate-induced colitis in mice. InternationalImmunopharmacology, 2002, (2):565-78
[20] Takei Y, Kunikata T, Aga M, et al. Tryptanthrin inhibits interferon-gamma production by Peyer's patch lymphocytes derived from mice that had been orally administered staphylococcal enterotoxin. Biol Pharm Bull. 2003,26(3): 365-7
[21] Danz H, Stoyanova S, Wippich P, et al. Identification and isolation of the cyclooxygenase-2 inhibitory principle in Isatis tinctoria [J]. Planta Med. 2001, 67 (5):411-6
[22] Danz H, Stoyanova S, Thomet OA, et al. Inhibitory activity of tryptanthrin on prostaglandin and leukotriene synthesis[J]. Planta Med. 2002,68(10):875-80
[23]王振义.白血病的诱导凋亡疗法[N].中国医学论坛报,2003,29(1):72-5
[24] Kimoto T, Hino K, Koya-Miyata S, et al. Cell differentiation and apoptosis of monocytic and promyelocytic leukemia cells (U-937 and HL-60) by tryptanthrin, an active ingredient of Polygonum tinctorium Lour [J]. Pathology international, 2001, 51(5):315-25
[25] Mu Ben-zhefu. A new cytotoxic active principle isolated from Polygonum tinctorium. Natural Medicine, 1999, 53(2):72-9
[26] Motoki T, Takami Y, Yagi Y, et al. Inhibition of hepatocyte growth factor induction in human dermal fibroblasts by tryptanthrin. Biol Pharm Bull, 2005, 28(2):260-6
[27] Sharma VM, Prasanna P, Seshu KV, et al. Novel indolo [2,1-b]quinazoline analogues as cytostatic agents: synthesis, biological evaluation and structure- activity relationship. Bioorg Med Chem Lett. 2002,12(17):2303-7
[28] Sung-Tsai Yu, Tzer-Ming Chen, Shih-Yun Tseng, et al. Tryptanthrin inhibits MDR1 and reverses doxorubicin resistance in breast cancer cells. Biochemical and Biophysical Research Communications. 2007, 358:79-84
[29] Koya-Miyata S, Kimoto T, Micallef MJ, et al. Prevention of azoxymethane- induced intestinal tumors by a crude ethyl acetate-extract and tryptanthrin extracted from Polygonum tinctorium Lour [J]. Anti-cancer Research, 2001,21(5):3295-300
[30] Bhattacharjee AK, Hartell MG, Nichols DA, et al. Structure-activity relationship study of antimalarial indolo [2,1-b]quinazoline-6,12-diones (tryptanthrins). Three dimensional harmacophore modeling and identification of new antimalarial candidates. Eur J Med Chem. 2004,39(1):59-67
[31] Bhattacharjee AK, Skanchy DJ, Jennings B, et al. Analysis of stereo- electronic properties, mechanism of action and pharmacophore of synthetic indolo[2,1-b] quinazoline-6,12-dione derivatives in relation to antileishmanial activity using quantum chemical, cyclic voltammetry and 3-D-Qsar catalyst procedures. J. Bioorg Med Chem. 2002,10: 1979-89
[32] Scovill J, Blank E, Konnick M, et al. Antitrypanosomal activities of tryptanthrins. Antimicrob Agents Chemother. 2002, 46(3):882-3
[33] Green DR, Reed JC. Mitochondria and apoptosis [J]. Science, 1998, 281(5381): 1309-12.
[34] Glaser T, Weller M. Caspase-dependent chemotherapy induced death of glioma cells requires mitochondrial cytochrome c release [J]. Biophys Res Commun, 2001, 281 (2): 322-7.
[35] Newmeyer D, Ferguson MS. Mitochondria: releasing power for life and unleashing the machineries of death [J]. Cell, 2003, 112 (4): 481-90.
[36] Armstrong JS. Mitochondrial membrane permeabilization: the sine quanon for cell death [J]. Bioessays, 2006, 28 (3): 253-60.
[37] Zamzami N, Kroemer G. The mitochondrion in apoptosis: how Pandora’s box opens [J]. Nat Rev Mol Cell Biol, 2001, 2 (1): 67-71.
[38] Saris NE, Carafoli E. A historical review of cellular calcium handling with emphasis on mitochondria [J]. Biochemistry (Moscow), 2005, 70(2): 187-94.
[39] Linda E, Frank AE, Kruyt E, et al. Cell death independent of caspases: A review [J]. Clin Cancer Res, 2005, 11(9): 3155-62.
[40] Scorrano L, Ashiya M, Buttle K, et al. A distinct pathway remodelsmitochondrial cristae and mobilizes cytochrome c during apoptosis [J]. Dev Cell, 2002, 2 (1): 55-67.
[41] Loo G, Saelens X, Gurp M, et al. The role of mitochondrial factors in apoptosis: a russian roulette with more than one bullet [J]. Cell Death Differ, 2002, 9 (10):1031-42.
[42] Hatai T, Matsuzawa A, Inoshita S, et al. Execution of apoptosis signal- regulating kinase1 (ASK1) induced apoptosis by the mitochondria- dependent caspase activation [J]. J Biol Chem, 2000, 275 (34): 26576-81.
[43] Green D R. Apoptotic pathways: the roads to ruin [J]. Cell, 1998, 94 (6): 695-8.
[44] Chang H Y, Yang X. Proteases for cell suicide: functions and regulation of caspases [J]. Microbiol Mol Biol Rev, 2000, 64 (4): 821-46
[45] Hsu SY, Hsueh AJ. Tissue-specific Bcl-2 protein partners in apoptosis: An ovarian paradigm [J]. Physiol Rev, 2000, 80 (2): 593-614.
[46] Schindler CK, Shinoda S, Simon RP, et al. Subcellular distribution of Bcl-2 family proteins and 14-3-3 within the hippocampus during seizure-induced neuronal death in the rat [J]. Neurosci Lett, 2004, 356(3): 163-6.
[47] Gross A, McDonnell JM, Korsmeyer SJ. Bcl-2 family members and the mitochondria in apoptosis [J]. Genes Dev, 1999, 13(15): 1899-911.
[48] Burlacu A. Regulation of apoptosis by Bcl-2 family proteins [J]. J Cell Mol Med, 2003, 7 (3): 249-57.
[49] Puthalakath H, Strasser A. Keeping killers on a tight leash: transcriptional and post-translational control of the pro-apoptotic activity of BH3-only proteins [J]. Cell Death Differ, 2002, 9 (5): 505-12.
[50] Tsujimoto Y. Cell death regulation by the Bcl-2 protein family in the mitoehondria [J]. J Cell Physiol, 2003, 195 (2): 158-67.
[51] Yin XM, Oltvai ZN, Korsmeyer SJ. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax [J]. Nature, 1994, 369 (6478): 321-3.
[52] Sattler M, Liang H, Nettesheim D, et al. Structure of Bcl-xL-Bak peptide complex: recognition between regulators of apoptosis [J]. Science, 1997, 275 (5302): 983-6.
[53] Petros AM, Nettesheim DG, Wang Y, et al. Rationale for Bcl-xL/Bad peptide complex formation from structure, mutagenesis and biophysical studies [J]. Protein Sci, 2000, 9(12): 2528-34.
[54] Orrenius S. Mitochondrial regulation of apoptotic cell death [J]. Toxicol Lett, 2004, 149 (123): 19-23.
[55] Chou JJ, Li H, Salvesen GS, et al. Solution structure of BID, an intracellular amplifier of apoptotic signaling [J]. Cell, 1999,96 (5): 615-24.
[56] Kuwana T, Mackey MR, Perkins G, et al. Bid, Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane [J]. Cell, 2002, 111(3): 331-42.
[57] Saito M, Korsmeyer SJ, Schlesinger PH. Bax-dependent transport of cytochrome c reconstituted in pure liposomes [J]. Nat Cell Biol, 2000, 2(8): 553-5.
[58] Haeberlein SL.Mitochondrial functions in apoptotic neuronal cell death [J]. Neurochem Res, 2004, 29(3): 521-30.
[59] Wu C, Fujihara H, Yao J, et al. Different expression patterns of Bcl-2, Bcl-xl, and Bax proteins after sublethal forebrain ischemia in C57Black/Crj6 mouse striatum [J]. Stroke, 2003, 34(7): 1803-8.
[60] Susin SA, Lorenzo HK, ZamzamiN, et al. Molecular characterization of mitochondrial apoptosis inducing factor [J]. Nature, 1999, 39 (7): 441-6.
[61] Skulachev VP. Cytochrome c in the apoptotic and antioxidant cascades [J]. FEBS Lett, 1998, 423 (3): 275-80.
[62] Vieira H, Kroemer G. Mitochondria as targets of apoptosis regulation by nitric oxide [J]. IUBMB Life, 2003, 55(10211): 613-20.
[63] Li K, Li Y, Shelton JM, et al. Cytochrome c deficiency causes embryonic lethality and attenuates stress-induced apoptosis [J]. Cell, 2000, 101(4): 389-9.
[64] Bras M,Queenan B, Susin SA. Programmed cell death via mitochondria:different modes of dying [J]. Biochemistry (Moscow), 2005, 70(2): 231-9.
[65] Rice AM, Wood JA, Milrose CG, et al. Conditions that enable human hematopoietic stem cell engraftment in all NOD-SCID mice [J]. Transplantation, 2000, 69 (5): 927-35.
[66] Chargui J, Moya MJ, Sanhadji K, et al. Anti-NK antibodies injected into recipient mice enhance engraftment and chimerism after allogeneic transplantation of fetal liver stem cells [J]. Thymus, 1997, 24 (4):233-46.
[67] Kollet O, Peled A, Byk T, et al . B-Microglobulin-deficient (B-mnull) NOD/SCID mice are excellent recipients for studying human stem cell function [J]. Blood, 2000, 95(10):3102-5.
[68] Ohsugi T, Yamaguchi K, Kumasaka T, et al. Rapid tumor death model for evaluation of new therapeutic agents for adult T-cell leukemia [J]. Lab Invest, 2004,84(2):263-6.
[69] Feuring-Buske M, Gerhard B, Cashman J, et al. Improved engraftment of human acute myeloid leukemia progenitor cells in beta 2-micro- globulin-deficient NOD/SCID mice transgenic for human growth factors[J].Leukemia, 2003,17(4):760-3.
[70] Mitchell P, Lee FT, Hall C, et al. Targeting primary human Ph(+) B-cell precursor leukemia engrafted SCID mice using radiolabeled anti-CD19 monoclonal antibodies[J]. J Nucl Med, 2003, 44 (7):1105-16.
[71] Dazzi F, Hasserjian R, Gordon MY, et al. Normal and chronic phase CML hematopoietic cells repopulate NOD/SCID bone marrow with different kinetics and cell lineage representation[J]. Hematol J, 2000,1(5):307-15.
[72] Peled A, Hardan I, Trakhtenbrot L, et al. Immature leukemic CD34+CX- CR4+ cells from CML patients have lower integrin-dependent migration and adhesion in response to the chemokine SDF-1[J]. Stem Cells, 2002, 20(3):259-66.
[73] Ramirez M, Diaz MA, Madero L, et al. Transplantation of marrow cells from children with standard risk-acute lymphoblastic leukemia at the endof therapy into NOD/SCID mice for detecting residual leukemic cells with in vivo growth potential [J]. Leuk Res, 2003, 27(12): 1153-7.
[74] Herrera L, Yarbrough S, Ghetie V, et al. Treatment of SCID/human Bcell precursor ALL with anti-CD19 and anti-CD22 immunotoxins [J]. Leukemia, 2003, 17(2):334-338.
[75] Golay J, Di Gaetano N, Amico D, et al. Gemtuzumab ozogamicin (Mylotarg) has therapeutic activity against CD33 acute lymphoblastic leukaemias in vitro and in vivo [J]. Br J Haematol, 2005, 128(3):310-7.
[76] Nijmeijer BA, Willemze R. An animal model for human cellular immunotherapy: specific eradication of human acute lymphoblastic leukemia by cytotoxic T lymphocytes in NOD/SCID mice [J]. Blood, 2002, 100 (2):654-7.
[77] Honma Y, Ishii Y, Sassa T, et al. Treatment of human promyelocytic leukemia in the SCID mouse model with cotylenin A, an inducer of myelomonocytic differentiation of leukemia cells[J]. Leuk Res, 2003, 27(11):1019-25.
[78] Levis M, Murphy KM, Pham R, et al. Internal tandem duplications of the FLT3 gene are present in leukemia stem cells [J]. Blood, 2005, 106(2): 673-80.
[79] Piloto O, Levis M, Huso D, et al. Inhibitory anti-FLT3 antibodies are capable of mediating antibody-dependent cell-mediated cytotoxicity and reducing engraftment of acute myelogenous leukemia blasts in nonobese diabetic/severe combined immunodeficient mice [J]. Cancer Res, 2005, 65(4):1514-35.
[80] Bai L, Kon K, Tatsumi M, et al. A human B-cell model established by transplantation of JOK-1 cells into SCID mice and an anti-leukemia efficacy of fludarabine phosphate [J]. Oncol Rep, 2000, 7(1):33-8.
[81] Zhang Y, Dawson MI, Mohammsd R, et al. Indeuction of apoptosis of human B-CLL and ALL cells by a novel retinoid and its nonretinoidalanalog [J].Blood, 2002, 100(8):2917-25.
[82] Garattini E, Parrella E, Diomede L, et al. ST1926, a novel and orally active retinoid-related molecule inducing apoptosis in myeloid leukemia cells: modulation of intracellular calcium homeostasis [J]. Blood, 2004, 103(1):194-207.
[83] Kerr JFR, Wyllie, Currie AR. Apoptosis, a basic biologic phenomenon with wide implication in tissue kinetics.Brit. J.Cancer. 1972, 26: 239-45
[84] YANG J, LIU X, BHALLA K, etal. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked [J]. Seienee, 1997, 275(5303): 1129-32
[85] Gorczyca W, Gong J, Ardelt B, et al. The cell cycle related differences in susceptilility of HL-60 cells to apoptosis induced by various antitumor agents. Cancer Res. 1993,53:3186-92
[86] Sun MG, Williams J, Munoz-Pinedo C, Perkins GA, Brown JM, Ellisman MH, Green DR, Frey TG. Correlated three-dimensional light and electron microscopy reveals transformation of mitochondria during apoptosis. Nat Cell Biol 2007, 9:1057-65
[87] Sareen D, Darjatmoko SR, Albert DM, Polans AS. Mitochondria, calcium, and calpain are key mediators of resveratrol-induced apoptosis in breast cancer. Mol Pharmacol 2007, 72: 1466-75
[88] Hortelano, S. and Bosca, L. 6-Mercptopurine decreases the Bcl-2/Bax ratio and induces apoptosis in activated splenic B lymphocytes. Mol. Pharmcol. 1997, 51, 414-21.
[89] Oltvai, Z.N., Milliman, C.L. and Korsmeyer, S.J. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell, 1993, 74: 609-19.
[90] Green DR, Reed JC.Mitochondria and apoptosis. Science.1998, 281(5381): 1309
[91] Brener C, Cadiou H, Vieira HL, et al.Bcl-2 and Bax regulate the channelactivity of the mitochondrial adenine nucleotide translocator. Oncogene, 2000, 19(3): 329-36
[92] Shimizu S, Narita M, Tsujimoto Y. Bcl-2 family proteins regulate the release of apopotogenic cytochrome c by the mitochondrial channel VDAC. Nature, 1999, 399(6735): 483-7
[93] Shimiz S, Ide T, Yanagida T, et al. Electrophysiological study of a novel large pore formed by Bax and the voltage-dependent anion channel that is permeable to cytochrome c.[J] Biol Chem, 2000, 275(16): 12321-5
[94] Li P, Nijhawan D, Budihardjo I, et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell,1997, 91(4):479-89
[95]缪珊,张海,石小鹏,孙纪元,周宣暄,谢艳华,王捷频,苗青,王四旺.色胺酮对人白血病细胞株K562细胞增殖抑制、凋亡诱导的影响.中国药理学通报. 2009,25(2):256-8
[96]陈奇主编.中药药理实验方法北京人民卫生出版社1994:206
[97]沈志祥,陈钰,李军民编著.简明临床血液病学.上海:上海科学技术文献出版社. 2002. 6:8,506
[98] Walter DH, Haendeler J, Galle J, et al. Cyclosporin A inhibits apoptosis of human endothelialcells by preventing release of cytochrome c from mitochondria [J]. Circulation, 1998, 98: 1153
[99] Leckie BJ. Targeting the renin-angiotensin system: what’s new. [J] Curr Med Chem Cardiovasc Hematol Agents, 2005, 3(1): 23-32.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |