胡桃醌抗肿瘤作用及其机制研究
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摘要
研究背景
恶性肿瘤是当前危害人类健康的重要疾病之一,是新世纪人类第一杀手,对人类生存构成最严重的威胁。目前较为成熟的治疗方法有手术治疗、放射治疗、药物治疗及免疫治疗四类。近几年来,肿瘤化疗取得了很大的进步,但对危害人类生命健康最严重的、占恶性肿瘤90%以上的实体瘤的治疗效果不尽人意;并且绝大多数抗肿瘤药物在抑制或杀伤肿瘤细胞的同时,对正常组织、器官不可避免地产生损害或毒性作用,给患者带来痛苦,甚至导致死亡。因此,开发与研究新型有效的抗肿瘤药物是生物医药科研领域的重大课题和长期任务。
我国的药用植物物种资源丰富,含抗癌活性成分的物种有200种左右,迄今已通过肿瘤细胞株及动物移植性肿瘤筛选的植物单体成分已超过4000余种。植物来源药物在化学结构和作用机制方面均具有多样性;同时,长期以来中医治疗肿瘤的研究已积累丰富的资料和经验,有助于开发和研制植物来源的抗肿瘤新药。因此,从天然药物中获取有效成分并以此为先导化合物是抗肿瘤药物研究和开发的重要途径之一。
胡桃醌(Juglone;Nucin)又名5-羟基-1,4-萘醌;5-羟基-1,4-萘二酮。是从胡桃楸新鲜根皮、枝皮、青果皮中分离出来的羟基萘醌类化合物,是胡桃楸中主要的毒性物质。在中国胡桃楸皮作为民间抗癌验方,历史悠久,作为胡桃楸的主要成分胡桃醌的抗癌活性研究也有报道。近年来有研究显示其具有抗真菌、抗衣原体或抗HIV病毒、抗癌、降血糖和杀虫等生物活性,越来越受到人们的重视。因此,进一步开发胡桃醌抗肿瘤活性,了解其作用机制,有着非常重要的现实意义。
目的
筛选敏感细胞株,探讨胡桃醌抗肿瘤作用的细胞学机制;在体实验考察胡桃醌抗肿瘤活性,并对其毒性进行评价;研究胡桃醌对肿瘤转移形成的影响;检测胡桃醌SD大鼠血浆蛋白结合率,为其机体代谢提供一定理论基础。
方法
1.采用MTT法测定胡桃醌对不同细胞株的生长抑制率,筛选敏感细胞株。
2.采用Flou-3/AM法检测胡桃醌对BEL-7402细胞内游离钙离子浓度的影响,通过流式细胞仪观察12.5μmol/L胡桃醌作用后细胞线粒体膜电势的变化、细胞周期分布及凋亡细胞比率,采用荧光偏振技术离体动态地检测30min内胡桃醌对BEL-7402细胞各向异性r、荧光偏振度mP和微粘度η的影响,分析细胞膜流动性的改变。
3.12.5μmol/L胡桃醌作用于BEL-7402细胞24h后固定,分别行HE染色、考马斯亮蓝染色和扫描电镜及透射电镜的样品制备。
4.200μmol/L、100μmol/L、50μmol/L、25μmol/L和12.5μmol/L胡桃醌作用于EAHY.926细胞株24h后MTT法检测细胞存活率,选用划痕法探讨胡桃醌对内皮细胞迁移的影响。采用大鼠主动脉无血清培养法,加入100μmol/L、50μmol/L、25μmol/L和12.5μmol/L浓度的胡桃醌,观察其对血管内皮细胞爬行及其微血管结构形成的影响;选用7日龄鸡胚进行鸡胚绒毛尿囊膜血管生成实验,探讨胡桃醌对新生血管形成的影响。
5.采用人肝癌BEL-7402细胞裸鼠皮下移植瘤模型,通过腹腔注射和局部注射两个给药途径进行胡桃醌药效学的研究。
6.采用小鼠B16/F10黑色素瘤人工肺转移模型研究4.5mg/kg、3mg/kg、1.5mg/kg和0.75mg/kg胡桃醌对肿瘤细胞血道转移的作用。
7.采用超滤法,通过高效液相(HPLC)考察胡桃醌的血浆蛋白结合率。
结果
1.不同类型的细胞株对胡桃醌的敏感性不同:人肝癌HepG2细胞株IC_(50)为10.93841μmol/L,人肝癌BEL-7402细胞株IC_(50)为14.66485μmol/L,人乳腺癌MCF-7细胞株IC_(50)为20.788μmol/L,人肺癌A549细胞株IC_(50)为24.41432μmol/L,人鼻咽癌CNE2细胞株IC_(50)为25.3178μmol/L,人宫颈癌HeLa细胞株IC_(50)为27.90769μmol/L,。
2.100μmol/L、50μmol/L、25μmol/L、12.5μmol/L、6.25μmol/L胡桃醌均可抑制人肝癌BEL-7402细胞的生长,诱导其凋亡,IC_(50)为13.78μmol/L。25μmol/L、12.5μmol/L胡桃醌作用BEL-7402细胞株24h后行PI染色,结果显示有明显的凋亡峰出现;同时存在明显的细胞碎片峰。而胡桃醌作用HepG2细胞株24h后行PI染色,结果显示只有明显的细胞碎片峰,而无凋亡峰出现。胡桃醌作用于BEL-7402细胞4h后线粒体膜电势降低,提示BEL-7402细胞已经开始凋亡级联反应。400μmol/L、200μmol/L、100μmol/L、50μmol/L、25μmol/L、12.5μmol/L、6.25μmol/L胡桃醌均可引起BEL-7402细胞膜的r、mP和η值显著性降低(P＜0.05),尤其在最初1min内r、mP、η值下降迅速,1min后各指标下降的趋势渐渐平缓。不同浓度的胡桃醌作用BEL-7402细胞30min后未见胞内Ca~(2+)浓度发生改变,结果提示胡桃醌可能通过Capase途径诱导细胞调亡。
3.12.5μmol/L胡桃醌作用BEL-7402细胞24h后细胞形态和细胞骨架均发生改变。扫描电镜结果显示BEL-7402细胞胞体体积缩小,与周围细胞群脱离,表面微绒毛卷曲、畸变、小球结构逐渐增多,细胞间连接逐渐断裂,细胞间隙增宽,有凋亡小体形成。透射电镜结果显示,细胞微绒毛消失,膜完整,胞核浓集,异染色质增多,内质网扩张,线粒体疏散、轻度肿胀,嵴消失。
4.200μmol/L、100μmol/L和50μmol/L浓度胡桃醌对EAHY.926有明显的抑制作用(P＜0.05),IC_(50)为122.848μmol/L;25μmol/L、12.5μmol/L和6.25μmol/L的胡桃醌促进EAHY.926细胞株的生长(P＜0.05)。1 00μmol/L、50μmol/L和25μmol/L的胡桃醌均可抑制大鼠主动脉微血管形成(P＜0.01),12.5μmol/L的胡桃醌可明显减少内皮细胞的爬行数量,延缓其爬行速度(P＜0.01)。200μmol/L、100μmol/L、50μmol/L及25μmol/L胡桃醌作用于鸡胚绒毛尿囊膜,其血管呈现现不同程度的充血、出血,甚至凝血,呈剂量依赖性;各组胡桃醌作用区域均无新生血管形成。
5.以600μg/kg、300μg/kg和150μg/kg胡桃醌腹腔注射于人肝癌BEL-7402细胞裸鼠皮下移植瘤模型,NK细胞活性检测发现,600μg/kg、300μg/kg胡桃醌组裸鼠NK细胞活性较正常对照组低(P均＜0.01),150μg/kg胡桃醌组的NK细胞活性与正常对照组无显著差异(P＞0.05);但600μg/kg胡桃醌组NK细胞活性与阳性对照组(5-Fu)相似(P＞0.05)。结果提示胡桃醌对小鼠免疫系统有一定的损伤作用。但600μg/kg、300μg/kg和150μg/kg胡桃醌对肿瘤生长没有明显的影响(P均＞0.05)。
不同PH(PH=7.4、PH=4.0)600μg/kg、300μg/kg和150μg/kg胡桃醌对人肝癌BEL-7402细胞裸鼠皮下移植瘤模型局部给药,结果发现不同PH值的600μg/kg、300μg/kg胡桃醌局部注射抑瘤作用与空白对照组相比存在显著差异(P均＜0.05),而与阳性对照组(5-Fu)组相比无显著差异(P＞0.05);同一浓度不同PH胡桃醌间的抑瘤作用无显著差异(P＞0.05)。
以4.5mg/kg、3mg/kg和1.5mg/kg胡桃醌腹腔注射于人肝癌BEL-7402细胞裸鼠皮下移植瘤模型,结果发现4.5mg/kg、3mg/kg、1.5mg/kg胡桃醌的肿瘤抑制率分别为78.24%、66.57%、48.94%;4.5mg/kg、3mg/kg胡桃醌的抑瘤作用可与阳性对照组相当(P均＞0.05)。但4.5mg/kg胡桃醌组裸鼠出现明显的皮下脂肪减少、消瘦,并有死亡现象。
6.4.5mg/kg、3mg/kg、1.5mg/kg和0.75mg/kg胡桃醌对小鼠黑色素瘤B16F10血道转移抑制率分别为27.30%、55.35%、31.52%和25.34%;3mg/kg胡桃醌抑瘤率与阳性对照组相当(P＞0.05)。而4.5mg/kg胡桃醌抑制率低可能是由于该剂量胡桃醌降低机体免疫功能造成的。
7.胡桃醌SD大鼠血浆蛋白结合率为90.92%。
结论
1.胡桃醌能有效抑制人源肿瘤细胞增殖;但细胞株类型不同,其敏感性不同。
2.胡桃醌能有效改变人肝癌BEL-7402细胞亚显微结构,诱导细胞死亡。
3.胡桃醌能显著增加BEL-7402细胞的膜流动性,促进药物进入细胞内部,提高药物对细胞的毒性。
4.胡桃醌具有抗血管生成作用。
5.胡桃醌可抑制小鼠黑色素瘤B16F10血道转移。
6.胡桃醌可抑制人肝癌BEL-7402细胞裸鼠皮下移植瘤的生长。
7.胡桃醌血浆蛋白结合率高,导致其需较高浓度才能发挥其抗肿瘤效果。
Malignant tumor is one of diseases which jeopardizes the human health and is the first killer to mankind in the new century.At present,more sophisticated treatments of malignant tumor includes surgery treatment,radiation therapy, pharmacotherapy and immunotherapy.And the cancer chemotherapy has made considerable progress,but the treatment of malignant solid tumor,one of the most serious diseases endangering human health and life,accounting for more than 90%of malignant tumors,failed to achieve satisfaction and the vast majority of anticancer drugs in inhibiting or killing tumor cells may induce destruction of normal tissues, inevitable damage of organs or other toxicity to the patients.Therefore,the discovery and study of new effective anti-cancer drugs is the major issue and a long-term task for the biomedical research.
Germplasm of medicinal plants in our country is rich in resources,which containing the active ingredient of Chinese herbal anti-cancer has about 200 kinds,and confirmed by transplanted animal tumor cell lines in vivo and in vitro, more than 4000 kinds of plant composition have antitumor activity.The chemical structure and the mechanisms of drugs from plant sources has the diversity.At the same time,a wealth of information and experience have been accumulated in a long-term study on chinese medicine treatment for tumor,which contribute to the discovery and development of plant sources of anti-tumor drugs.Therefore,to obtain drugs from natural ingredients and to make it the lead compounds are one of the important ways for research and development of anti-cancer drugs.
Juglone(Nucin),is known as 5 - hydroxy -1,4 - naphthoquinone,5 - hydroxy -1,4 - naphthalene dione.Being the main toxic substances of juglans,it is separated from the root bark,branch bark,green peel of Juglans mandshurica and belongs to the hydroxy-naphthoquinone compounds.Juglans were used as a non-governmental anti-cancer walnut recipes in China for a long history.Juglone,as the main component of Juglans mandshurica,its anti-cancer activity has been reported.In recent years, some studies have showed juglone have various biological activities,such as anti-fungal,anti-Chlamydia or HIV virus,anti-cancer,lowering blood glucose and pesticides,and has been paid more and more attention.Therefore,further developing anti-tumor activity of juglone and understanding its mechanism has a very important practical significance.
Objective
To screen sensitive cell lines and learn the anti-cancer mechanism of junlone.To evaluate the inhibitory effect and toxicity of juglone and investigate the inhibitory effect of juglone on the tumor metastasis.To study the plasma protein binding rate of juglone.
Methods
1.Rates of growth inhibition of juglone on different cell lines were obstained by MTT method.
2.BEL-7402 cells were treated with juglone at different concentration in vitro,probed with Flow-3/AM and the intracellular calcium concentrations were detected.Flow cytometry was used to observe cell apoptosis and mitochondrial membrane potential changes.The fluorescence depolarization method was used to measure values of fluorescence anisotropy,fluorescence polarization as well as microviscosity in BEL-7402 cells and microviscosity in liposome continuously for 30 min.
3.BEL-7402 cell were incubated with 12.5μmol/Ljuglone for 24 hours,and fixed in 2.5%glutaraldehyde for HE,coomassie brilliant blue staining,scanning electron microscope and transmission electron microscopy observation.
4.The effect of a series of concentration of juglone on the growth of EAHY.926 were measured by MTT.Serum-free culture of the rat aorta was conducted,with 100μmol/L,50μmol/L,25μmol/L and 12.5μmol/L dosages of juglone added,to observe its effect on crawling of the vascular endothelial cells and formation of capillary structure.7-day-old chick embryoes were selected for experimental angiogenesis model to explore the impact of juglone on neovascularization.
5.Human hepatic carcinoma BEL-7402 cells were injected into nude mice subcutaneously.The resultant tumor were taken and prepared into small tissue blocks,which were implanted subcutaneously into nude mice to establish mouse models bearing human hepatoma.Different doses of juglone were administatored by intraperitoneal and local injection.The general condition of the tumor-bearing mice and the growth of the tumors were observed for pharmacodynamic study of quinone.
6.B16-F10 cells were injected into the tail veins of C57BL/6 mice to establish an experimental lung metastasis model,and the effect of juglone on lung metastasis was observed.
7.The ultrafiltration was employed to determine the plasma protein binding rate of juglone.The plasma concentrations of juglone were measured by HPLC.
Results
1.Sensitivities of different cell lines to juglone were different:IC_(50) of human hepatocellular carcinoma BEL-7402 cell line,Human lung cancer A549 cell line,Human breast cancer MCF-7 cell line,human nasopharyngeal carcinoma CNE2 cell line,human cervical carcinoma Hela cell line,human hepatocellular carcinoma HepG2 cell line were 14.664μol/L,24.414μmol/L,20.788μmol/L,25.317μmol/L, 27.907μmol/L,10.938μmol/L,respectively.
2.The results showed that juglone at different dosages could inhibit the growth of human liver cancer BEL-7402,and induce cell apoptosis,IC_(50) was 13.78μmol/L.Juglone induced a significant decrease of fluorescence anisotropy, fluorescence polarization as well as microviscosity in BEL-7402 cells (P＜0.05),particularly in the first 1 min.After 1 min,the values of anisotropy were remained lower levels.There was no change of intracellular Ca~(2+) concentration of BEL-7402 cells with juglone for 30min.The results suggested that juglone may induce apoptosis through Capase.
3.It was found that changes developed in the cytomorphology and cytoskeleton of these cells.According to the scanning electron microscope,we found that the volume of cell bodies were decreased,microvilli on cell surface crimped and aberrated. intercellular junction was ruptured and the intercellular space was enlarged.
4.We found that juglone of 200μmol/L,100μmol/L and 50μmol/L could effectively inhibit the proliferation of EAHY.926 in a dose-dependent manner(P＜0.05),with an IC_(50) of 122.848μmol/L according to the results of MTT.While juglone of 25μmol/L,12.5μmol/L and 6.25μmol/L could promote growth of EAHY.926(P＜0.05 ). juglone of 100μmol/L,50μmol/L and 25μmol/L could completely inhibit the formation of new vessels,and juglone of 12.5μmol/L could reduce the number of endothelial cells and slow its crawling speed(P＜0.01).Juglone of 200μmol/L, 100μmol/L,50μmol/L and 25μmol/L have vascular stimulation,hemolyzation, agglutination as a dose-dependent manner and juglone could inhibit angiogenesis.
5.The inhibitory effect of juglone of 600μg/kg,300μg/kg and 150μg/kg on the growth of human hepatocellular carcinoma Bel-7402 in nude mice was studied.The results revealed that NK cell activity decreased in nude mice with juglone of 600μg/kg, 300μg/kg.Compared with the positive control group(5-Fu),NK cell activity of nude mice with juglone of 600ug/kg group was no significant difference(P＞0.05).Juglone of600μg/kg,300μg/kg and 150μg/kg had no significant impact on tumor growth.
Juglone of 600μg/kg,300μg/kg and 150μg/kg in PH=7.4 and PH=4.0 respectively were given by topical injection to human hepatocellular carcinoma BEL-7402 cells transplanted subcutaneously in nude models,we found 600μg/kg, 300μg/kg concentration juglone in different PH could effectly inhibit the growth of tumor and compared to the positive control group(5-Fu),there was no significant difference between all groups(P＞0.05),and the anti-tumor effect of the same concentration between different PH were no significant difference(P＞0.05).
Juglone of 4.5mg/kg,3mg/kg and 1.5mg/kg were given by intraperitoneal injection to human liver cancer BEL-7402 cells transplanted subcutaneously in nude model,the results showed that the juglone could effectly inhibit the tumor growth, the tumor inhibitory rate of juglone of 4.5mg/kg,3.0 mg/kg,1.5 mg/kg were 78.24%,66.57%and 48.94%respectively.4.5mg/kg and 3mg/kg juglone had the same anti-tumer effect when compared with the positive control group(P＞0.05,P＞0.05). But there were significantly reduced subcutaneous fat,weight loss,and death cases in 4.5 mg/kg juglone group.
6.The number of metastases in the animals that were given juglone of 4.5mg/kg,3mg/kg,1.5mg/kg and 7.5mg/kg was significantly reduced as compared with the vehicle control(P＜0.05),and the inhibition rates were 27.30%,55.35%,31.52%,25.34%,respectively.Compared with the positive control group,the inhibitory rate of 3mg/kg juglone was no significant difference(P＞0.05). The inhibitory rate of 4.5mg/kg juglone are low probably because the juglone could decrease immune function of nude mice.
7.The plasma protein binding rate of juglone with SD rat plasma at the concentration of 100μg/ml was more than 90%.
Conclusion
1.Juglone could effectively inhibit proliferation of human origin cancer and sensitivities of different cell lines to juglone were different.
2.Juglone could significantly increase the membrane fluidity of BEL-7402 cells,and promote the drug to enter cells,enhance the toxicity of drugs on cells.
3.Juglone could effectively change the submicroscopic structure of human hepatocellular carcinoma BEL-7402 cells.
4.Juglone had the role of anti-angiogenesis.
5.Juglone could inhibit the growth of human liver cancer BEL-7402 cells in nude mice transplanted tumor,but there is a certain side effects,and the scope of drug safety were smaller.
6.Juglone could inhibit melanoma metastasis of B16F10.
7.The binding rate of juglone with SD rat plasma protein was so high that it would have anti-cancer effect in high doses.
恶性肿瘤是当前危害人类健康的重要疾病之一,是新世纪人类第一杀手,对人类生存构成最严重的威胁。目前较为成熟的治疗方法有手术治疗、放射治疗、药物治疗及免疫治疗四类。近几年来,肿瘤化疗取得了很大的进步,但对危害人类生命健康最严重的、占恶性肿瘤90%以上的实体瘤的治疗效果不尽人意;并且绝大多数抗肿瘤药物在抑制或杀伤肿瘤细胞的同时,对正常组织、器官不可避免地产生损害或毒性作用,给患者带来痛苦,甚至导致死亡。因此,开发与研究新型有效的抗肿瘤药物是生物医药科研领域的重大课题和长期任务。
我国的药用植物物种资源丰富,含抗癌活性成分的物种有200种左右,迄今已通过肿瘤细胞株及动物移植性肿瘤筛选的植物单体成分已超过4000余种。植物来源药物在化学结构和作用机制方面均具有多样性;同时,长期以来中医治疗肿瘤的研究已积累丰富的资料和经验,有助于开发和研制植物来源的抗肿瘤新药。因此,从天然药物中获取有效成分并以此为先导化合物是抗肿瘤药物研究和开发的重要途径之一。
胡桃醌(Juglone;Nucin)又名5-羟基-1,4-萘醌;5-羟基-1,4-萘二酮。是从胡桃楸新鲜根皮、枝皮、青果皮中分离出来的羟基萘醌类化合物,是胡桃楸中主要的毒性物质。在中国胡桃楸皮作为民间抗癌验方,历史悠久,作为胡桃楸的主要成分胡桃醌的抗癌活性研究也有报道。近年来有研究显示其具有抗真菌、抗衣原体或抗HIV病毒、抗癌、降血糖和杀虫等生物活性,越来越受到人们的重视。因此,进一步开发胡桃醌抗肿瘤活性,了解其作用机制,有着非常重要的现实意义。
目的
筛选敏感细胞株,探讨胡桃醌抗肿瘤作用的细胞学机制;在体实验考察胡桃醌抗肿瘤活性,并对其毒性进行评价;研究胡桃醌对肿瘤转移形成的影响;检测胡桃醌SD大鼠血浆蛋白结合率,为其机体代谢提供一定理论基础。
方法
1.采用MTT法测定胡桃醌对不同细胞株的生长抑制率,筛选敏感细胞株。
2.采用Flou-3/AM法检测胡桃醌对BEL-7402细胞内游离钙离子浓度的影响,通过流式细胞仪观察12.5μmol/L胡桃醌作用后细胞线粒体膜电势的变化、细胞周期分布及凋亡细胞比率,采用荧光偏振技术离体动态地检测30min内胡桃醌对BEL-7402细胞各向异性r、荧光偏振度mP和微粘度η的影响,分析细胞膜流动性的改变。
3.12.5μmol/L胡桃醌作用于BEL-7402细胞24h后固定,分别行HE染色、考马斯亮蓝染色和扫描电镜及透射电镜的样品制备。
4.200μmol/L、100μmol/L、50μmol/L、25μmol/L和12.5μmol/L胡桃醌作用于EAHY.926细胞株24h后MTT法检测细胞存活率,选用划痕法探讨胡桃醌对内皮细胞迁移的影响。采用大鼠主动脉无血清培养法,加入100μmol/L、50μmol/L、25μmol/L和12.5μmol/L浓度的胡桃醌,观察其对血管内皮细胞爬行及其微血管结构形成的影响;选用7日龄鸡胚进行鸡胚绒毛尿囊膜血管生成实验,探讨胡桃醌对新生血管形成的影响。
5.采用人肝癌BEL-7402细胞裸鼠皮下移植瘤模型,通过腹腔注射和局部注射两个给药途径进行胡桃醌药效学的研究。
6.采用小鼠B16/F10黑色素瘤人工肺转移模型研究4.5mg/kg、3mg/kg、1.5mg/kg和0.75mg/kg胡桃醌对肿瘤细胞血道转移的作用。
7.采用超滤法,通过高效液相(HPLC)考察胡桃醌的血浆蛋白结合率。
结果
1.不同类型的细胞株对胡桃醌的敏感性不同:人肝癌HepG2细胞株IC_(50)为10.93841μmol/L,人肝癌BEL-7402细胞株IC_(50)为14.66485μmol/L,人乳腺癌MCF-7细胞株IC_(50)为20.788μmol/L,人肺癌A549细胞株IC_(50)为24.41432μmol/L,人鼻咽癌CNE2细胞株IC_(50)为25.3178μmol/L,人宫颈癌HeLa细胞株IC_(50)为27.90769μmol/L,。
2.100μmol/L、50μmol/L、25μmol/L、12.5μmol/L、6.25μmol/L胡桃醌均可抑制人肝癌BEL-7402细胞的生长,诱导其凋亡,IC_(50)为13.78μmol/L。25μmol/L、12.5μmol/L胡桃醌作用BEL-7402细胞株24h后行PI染色,结果显示有明显的凋亡峰出现;同时存在明显的细胞碎片峰。而胡桃醌作用HepG2细胞株24h后行PI染色,结果显示只有明显的细胞碎片峰,而无凋亡峰出现。胡桃醌作用于BEL-7402细胞4h后线粒体膜电势降低,提示BEL-7402细胞已经开始凋亡级联反应。400μmol/L、200μmol/L、100μmol/L、50μmol/L、25μmol/L、12.5μmol/L、6.25μmol/L胡桃醌均可引起BEL-7402细胞膜的r、mP和η值显著性降低(P＜0.05),尤其在最初1min内r、mP、η值下降迅速,1min后各指标下降的趋势渐渐平缓。不同浓度的胡桃醌作用BEL-7402细胞30min后未见胞内Ca~(2+)浓度发生改变,结果提示胡桃醌可能通过Capase途径诱导细胞调亡。
3.12.5μmol/L胡桃醌作用BEL-7402细胞24h后细胞形态和细胞骨架均发生改变。扫描电镜结果显示BEL-7402细胞胞体体积缩小,与周围细胞群脱离,表面微绒毛卷曲、畸变、小球结构逐渐增多,细胞间连接逐渐断裂,细胞间隙增宽,有凋亡小体形成。透射电镜结果显示,细胞微绒毛消失,膜完整,胞核浓集,异染色质增多,内质网扩张,线粒体疏散、轻度肿胀,嵴消失。
4.200μmol/L、100μmol/L和50μmol/L浓度胡桃醌对EAHY.926有明显的抑制作用(P＜0.05),IC_(50)为122.848μmol/L;25μmol/L、12.5μmol/L和6.25μmol/L的胡桃醌促进EAHY.926细胞株的生长(P＜0.05)。1 00μmol/L、50μmol/L和25μmol/L的胡桃醌均可抑制大鼠主动脉微血管形成(P＜0.01),12.5μmol/L的胡桃醌可明显减少内皮细胞的爬行数量,延缓其爬行速度(P＜0.01)。200μmol/L、100μmol/L、50μmol/L及25μmol/L胡桃醌作用于鸡胚绒毛尿囊膜,其血管呈现现不同程度的充血、出血,甚至凝血,呈剂量依赖性;各组胡桃醌作用区域均无新生血管形成。
5.以600μg/kg、300μg/kg和150μg/kg胡桃醌腹腔注射于人肝癌BEL-7402细胞裸鼠皮下移植瘤模型,NK细胞活性检测发现,600μg/kg、300μg/kg胡桃醌组裸鼠NK细胞活性较正常对照组低(P均＜0.01),150μg/kg胡桃醌组的NK细胞活性与正常对照组无显著差异(P＞0.05);但600μg/kg胡桃醌组NK细胞活性与阳性对照组(5-Fu)相似(P＞0.05)。结果提示胡桃醌对小鼠免疫系统有一定的损伤作用。但600μg/kg、300μg/kg和150μg/kg胡桃醌对肿瘤生长没有明显的影响(P均＞0.05)。
不同PH(PH=7.4、PH=4.0)600μg/kg、300μg/kg和150μg/kg胡桃醌对人肝癌BEL-7402细胞裸鼠皮下移植瘤模型局部给药,结果发现不同PH值的600μg/kg、300μg/kg胡桃醌局部注射抑瘤作用与空白对照组相比存在显著差异(P均＜0.05),而与阳性对照组(5-Fu)组相比无显著差异(P＞0.05);同一浓度不同PH胡桃醌间的抑瘤作用无显著差异(P＞0.05)。
以4.5mg/kg、3mg/kg和1.5mg/kg胡桃醌腹腔注射于人肝癌BEL-7402细胞裸鼠皮下移植瘤模型,结果发现4.5mg/kg、3mg/kg、1.5mg/kg胡桃醌的肿瘤抑制率分别为78.24%、66.57%、48.94%;4.5mg/kg、3mg/kg胡桃醌的抑瘤作用可与阳性对照组相当(P均＞0.05)。但4.5mg/kg胡桃醌组裸鼠出现明显的皮下脂肪减少、消瘦,并有死亡现象。
6.4.5mg/kg、3mg/kg、1.5mg/kg和0.75mg/kg胡桃醌对小鼠黑色素瘤B16F10血道转移抑制率分别为27.30%、55.35%、31.52%和25.34%;3mg/kg胡桃醌抑瘤率与阳性对照组相当(P＞0.05)。而4.5mg/kg胡桃醌抑制率低可能是由于该剂量胡桃醌降低机体免疫功能造成的。
7.胡桃醌SD大鼠血浆蛋白结合率为90.92%。
结论
1.胡桃醌能有效抑制人源肿瘤细胞增殖;但细胞株类型不同,其敏感性不同。
2.胡桃醌能有效改变人肝癌BEL-7402细胞亚显微结构,诱导细胞死亡。
3.胡桃醌能显著增加BEL-7402细胞的膜流动性,促进药物进入细胞内部,提高药物对细胞的毒性。
4.胡桃醌具有抗血管生成作用。
5.胡桃醌可抑制小鼠黑色素瘤B16F10血道转移。
6.胡桃醌可抑制人肝癌BEL-7402细胞裸鼠皮下移植瘤的生长。
7.胡桃醌血浆蛋白结合率高,导致其需较高浓度才能发挥其抗肿瘤效果。
Malignant tumor is one of diseases which jeopardizes the human health and is the first killer to mankind in the new century.At present,more sophisticated treatments of malignant tumor includes surgery treatment,radiation therapy, pharmacotherapy and immunotherapy.And the cancer chemotherapy has made considerable progress,but the treatment of malignant solid tumor,one of the most serious diseases endangering human health and life,accounting for more than 90%of malignant tumors,failed to achieve satisfaction and the vast majority of anticancer drugs in inhibiting or killing tumor cells may induce destruction of normal tissues, inevitable damage of organs or other toxicity to the patients.Therefore,the discovery and study of new effective anti-cancer drugs is the major issue and a long-term task for the biomedical research.
Germplasm of medicinal plants in our country is rich in resources,which containing the active ingredient of Chinese herbal anti-cancer has about 200 kinds,and confirmed by transplanted animal tumor cell lines in vivo and in vitro, more than 4000 kinds of plant composition have antitumor activity.The chemical structure and the mechanisms of drugs from plant sources has the diversity.At the same time,a wealth of information and experience have been accumulated in a long-term study on chinese medicine treatment for tumor,which contribute to the discovery and development of plant sources of anti-tumor drugs.Therefore,to obtain drugs from natural ingredients and to make it the lead compounds are one of the important ways for research and development of anti-cancer drugs.
Juglone(Nucin),is known as 5 - hydroxy -1,4 - naphthoquinone,5 - hydroxy -1,4 - naphthalene dione.Being the main toxic substances of juglans,it is separated from the root bark,branch bark,green peel of Juglans mandshurica and belongs to the hydroxy-naphthoquinone compounds.Juglans were used as a non-governmental anti-cancer walnut recipes in China for a long history.Juglone,as the main component of Juglans mandshurica,its anti-cancer activity has been reported.In recent years, some studies have showed juglone have various biological activities,such as anti-fungal,anti-Chlamydia or HIV virus,anti-cancer,lowering blood glucose and pesticides,and has been paid more and more attention.Therefore,further developing anti-tumor activity of juglone and understanding its mechanism has a very important practical significance.
Objective
To screen sensitive cell lines and learn the anti-cancer mechanism of junlone.To evaluate the inhibitory effect and toxicity of juglone and investigate the inhibitory effect of juglone on the tumor metastasis.To study the plasma protein binding rate of juglone.
Methods
1.Rates of growth inhibition of juglone on different cell lines were obstained by MTT method.
2.BEL-7402 cells were treated with juglone at different concentration in vitro,probed with Flow-3/AM and the intracellular calcium concentrations were detected.Flow cytometry was used to observe cell apoptosis and mitochondrial membrane potential changes.The fluorescence depolarization method was used to measure values of fluorescence anisotropy,fluorescence polarization as well as microviscosity in BEL-7402 cells and microviscosity in liposome continuously for 30 min.
3.BEL-7402 cell were incubated with 12.5μmol/Ljuglone for 24 hours,and fixed in 2.5%glutaraldehyde for HE,coomassie brilliant blue staining,scanning electron microscope and transmission electron microscopy observation.
4.The effect of a series of concentration of juglone on the growth of EAHY.926 were measured by MTT.Serum-free culture of the rat aorta was conducted,with 100μmol/L,50μmol/L,25μmol/L and 12.5μmol/L dosages of juglone added,to observe its effect on crawling of the vascular endothelial cells and formation of capillary structure.7-day-old chick embryoes were selected for experimental angiogenesis model to explore the impact of juglone on neovascularization.
5.Human hepatic carcinoma BEL-7402 cells were injected into nude mice subcutaneously.The resultant tumor were taken and prepared into small tissue blocks,which were implanted subcutaneously into nude mice to establish mouse models bearing human hepatoma.Different doses of juglone were administatored by intraperitoneal and local injection.The general condition of the tumor-bearing mice and the growth of the tumors were observed for pharmacodynamic study of quinone.
6.B16-F10 cells were injected into the tail veins of C57BL/6 mice to establish an experimental lung metastasis model,and the effect of juglone on lung metastasis was observed.
7.The ultrafiltration was employed to determine the plasma protein binding rate of juglone.The plasma concentrations of juglone were measured by HPLC.
Results
1.Sensitivities of different cell lines to juglone were different:IC_(50) of human hepatocellular carcinoma BEL-7402 cell line,Human lung cancer A549 cell line,Human breast cancer MCF-7 cell line,human nasopharyngeal carcinoma CNE2 cell line,human cervical carcinoma Hela cell line,human hepatocellular carcinoma HepG2 cell line were 14.664μol/L,24.414μmol/L,20.788μmol/L,25.317μmol/L, 27.907μmol/L,10.938μmol/L,respectively.
2.The results showed that juglone at different dosages could inhibit the growth of human liver cancer BEL-7402,and induce cell apoptosis,IC_(50) was 13.78μmol/L.Juglone induced a significant decrease of fluorescence anisotropy, fluorescence polarization as well as microviscosity in BEL-7402 cells (P＜0.05),particularly in the first 1 min.After 1 min,the values of anisotropy were remained lower levels.There was no change of intracellular Ca~(2+) concentration of BEL-7402 cells with juglone for 30min.The results suggested that juglone may induce apoptosis through Capase.
3.It was found that changes developed in the cytomorphology and cytoskeleton of these cells.According to the scanning electron microscope,we found that the volume of cell bodies were decreased,microvilli on cell surface crimped and aberrated. intercellular junction was ruptured and the intercellular space was enlarged.
4.We found that juglone of 200μmol/L,100μmol/L and 50μmol/L could effectively inhibit the proliferation of EAHY.926 in a dose-dependent manner(P＜0.05),with an IC_(50) of 122.848μmol/L according to the results of MTT.While juglone of 25μmol/L,12.5μmol/L and 6.25μmol/L could promote growth of EAHY.926(P＜0.05 ). juglone of 100μmol/L,50μmol/L and 25μmol/L could completely inhibit the formation of new vessels,and juglone of 12.5μmol/L could reduce the number of endothelial cells and slow its crawling speed(P＜0.01).Juglone of 200μmol/L, 100μmol/L,50μmol/L and 25μmol/L have vascular stimulation,hemolyzation, agglutination as a dose-dependent manner and juglone could inhibit angiogenesis.
5.The inhibitory effect of juglone of 600μg/kg,300μg/kg and 150μg/kg on the growth of human hepatocellular carcinoma Bel-7402 in nude mice was studied.The results revealed that NK cell activity decreased in nude mice with juglone of 600μg/kg, 300μg/kg.Compared with the positive control group(5-Fu),NK cell activity of nude mice with juglone of 600ug/kg group was no significant difference(P＞0.05).Juglone of600μg/kg,300μg/kg and 150μg/kg had no significant impact on tumor growth.
Juglone of 600μg/kg,300μg/kg and 150μg/kg in PH=7.4 and PH=4.0 respectively were given by topical injection to human hepatocellular carcinoma BEL-7402 cells transplanted subcutaneously in nude models,we found 600μg/kg, 300μg/kg concentration juglone in different PH could effectly inhibit the growth of tumor and compared to the positive control group(5-Fu),there was no significant difference between all groups(P＞0.05),and the anti-tumor effect of the same concentration between different PH were no significant difference(P＞0.05).
Juglone of 4.5mg/kg,3mg/kg and 1.5mg/kg were given by intraperitoneal injection to human liver cancer BEL-7402 cells transplanted subcutaneously in nude model,the results showed that the juglone could effectly inhibit the tumor growth, the tumor inhibitory rate of juglone of 4.5mg/kg,3.0 mg/kg,1.5 mg/kg were 78.24%,66.57%and 48.94%respectively.4.5mg/kg and 3mg/kg juglone had the same anti-tumer effect when compared with the positive control group(P＞0.05,P＞0.05). But there were significantly reduced subcutaneous fat,weight loss,and death cases in 4.5 mg/kg juglone group.
6.The number of metastases in the animals that were given juglone of 4.5mg/kg,3mg/kg,1.5mg/kg and 7.5mg/kg was significantly reduced as compared with the vehicle control(P＜0.05),and the inhibition rates were 27.30%,55.35%,31.52%,25.34%,respectively.Compared with the positive control group,the inhibitory rate of 3mg/kg juglone was no significant difference(P＞0.05). The inhibitory rate of 4.5mg/kg juglone are low probably because the juglone could decrease immune function of nude mice.
7.The plasma protein binding rate of juglone with SD rat plasma at the concentration of 100μg/ml was more than 90%.
Conclusion
1.Juglone could effectively inhibit proliferation of human origin cancer and sensitivities of different cell lines to juglone were different.
2.Juglone could significantly increase the membrane fluidity of BEL-7402 cells,and promote the drug to enter cells,enhance the toxicity of drugs on cells.
3.Juglone could effectively change the submicroscopic structure of human hepatocellular carcinoma BEL-7402 cells.
4.Juglone had the role of anti-angiogenesis.
5.Juglone could inhibit the growth of human liver cancer BEL-7402 cells in nude mice transplanted tumor,but there is a certain side effects,and the scope of drug safety were smaller.
6.Juglone could inhibit melanoma metastasis of B16F10.
7.The binding rate of juglone with SD rat plasma protein was so high that it would have anti-cancer effect in high doses.
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