Recent Research and Progress of Juglone in Anti-cancer

Reporter: Jiao-lin Bao

Advisor: Maggie Pui Man, Hoi (Ph.D) (Cantab)

Pharmacology & Safety Evaluation

Content

Background

Objective

Results

Discussion

Methods

Juglans mandshurica Maxim walnut

What is Juglone (胡桃醌 ) ?

Clinical preparation:青龙衣注射剂、青龙衣粉针剂、复方青龙衣胶囊

Fig.1 The chemical structure of Juglone

Fig.2 Juglans mandshurica Maxim (核桃楸 )

Objective

1. To review the effect of juglone on the different cancer cells in recent studies.

2. To investigate the molecular mechanism of apoptosis induced by Juglone.

Search Strategy

Title： Juglone/胡桃醌 /核桃醌 /胡桃楸 /核桃楸 /青龙衣

Abstract： cancer /肿瘤 Juglans/ mandshurica

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References[1] JI YU-BIN Y B, QU Z Y, ZOU X. Juglone-induced apoptosis in human gastric cancer SGC-7901 cells

via the mitochondrial pathway [J]. Experimental and Toxicologic Pathology, 2011, 63(1-2): 69-78.

[2] KIRAN AITHAL B, SUNIL KUMAR M R, NAGESHWAR RAO B, et al. Juglone, a naphthoquinone from walnut, exerts cytotoxic and genotoxic effects against cultured melanoma tumor cells [J]. Cell Biology International, 2009, 33(10): 1039-49.

[3] LI L, CHEN P, LIAN H K, et al. Juglone inhibits the proliferation of human esophageal carcinoma EC1 cells [J]. World Chinese Journal of Digestology, 2010, 18(11): 1147-51.

[4] MONTENEGRO R C, ARA JO A J, MOLINA M T, et al. Cytotoxic activity of naphthoquinones with special emphasis on juglone and its 5-O-methyl derivative [J]. Chemico-Biological Interactions, 2010, 184(3): 439-48.

[5] PAULSEN M T, LJUNGMAN M. The natural toxin juglone causes degradation of p53 and induces rapid H2AX phosphorylation and cell death in human fibroblasts [J]. Toxicol Appl Pharmacol, 2005, 209(1): 1-9.

[6] POLONIK S G, PROKOF'EVA N G, AGAFONOVA I G, et al. Antitumor and immunostimulating activity of 5-hydroxy-1,4-naphthoquinone (juglone) o- and s-acetylglycosides [J]. Pharmaceutical Chemistry Journal, 2003, 37(8): 397-8.

[7] SUGIE S, OKAMOTO K, RAHMAN K M W, et al. Inhibitory effects of plumbagin and juglone on azoxymethane-induced intestinal carcinogenesis in rats [J]. Cancer Letters, 1998, 127(1-2): 177-83.

[8] XU H L, YU X F, QU S C, et al. Anti-proliferative effect of Juglone from Juglans mandshurica Maxim on human leukemia cell HL-60 by inducing apoptosis through the mitochondria-dependent pathway [J]. Eur J Pharmacol, 2010, 645(1-3): 14-22.

References[9] 陈丽 . 胡桃醌抗肿瘤作用及其机制研究 [D], 2009.

[10]李智博 . 胡桃楸活性成分分离解析及抗肿瘤作用的研究 [D], 2008.

[11]徐华丽 . 核桃醌诱导白血病 HL-60 细胞凋亡的作用及分子机制研究 [D], 2009.

[12]关健 . 胡桃醌体外诱导 LS-174T 细胞凋亡机制的初步研究 [D], 2010.

[13]姬艳菊 . 胡桃楸树皮提取物对人胃癌细胞 SGC-7901 作用的实验研究 [D], 2009.

[14]周媛媛 . 抗肿瘤中药青龙衣化学成分的研究 [D], 2008.

[15]陈丽 , 那顺巴雅尔 , 张建 , et al. 胡桃醌对人肝癌 BEL-7402 细胞亚显微结构的影响 [J]. 南方医科大学学报 , 2009, 06): 1208-11.

[16]陈丽 , 那顺巴雅尔 , 张建 , et al. 胡桃醌对人肝癌 BEL-7402 细胞增殖和膜流动性的影响 [J]. 数理医药学杂志 , 2009, 02): 144-9.

[17]崔玉强 . 核桃楸树皮化学成分及药物活性研究进展 [J]. 海峡药学 , 2010, 22(1):

[18]季宇彬 , 曲中原 , 邹翔 , et al. 青龙衣中胡桃醌对 S_(180) 肉瘤小鼠的抑瘤作用研究 [J]. 中国药学杂志 , 2009, 03): 195-9.

[19]孔陵生 , 邹翔 , 季宇彬 . 青龙衣中胡桃醌对 A-549 细胞周期及凋亡影响 [J]. 哈尔滨商业大学学报(自然科学版 ), 2008, 06): 641-4+77.

[20]雷涛 . 核桃楸皮抗肿瘤化学成分研究概述 [J]. 中国医药导报 , 2010, 07(23):

[21]刘艳萍 . 核桃楸化学成分及药理活性研究进展 [J]. 齐鲁药事 , 2010, 29(1):

[22]刘丹萍 , 张立钦 , 陈安良 , et al. 山核桃外果皮中胡桃醌含量测定及抑菌活性 [J]. 农药 , 2010, 09): 686-8+701.

[23]杨赛 . 胡桃楸树皮对红细胞氧化损伤抑制作用及其活性成分探讨 [J]. 现代预防医学 , 2007, 34(19):

Methods & Results

In vitro1. MTT assay, LDH assay----cell viability

2. Staining----Morphology (形态学观察 )

3. Flow cytometry(流式细胞仪 )percentage of apoptotic cells,mitochondrial membrane

potential and ROS

4. qPCR----gene changes

5. Western Blotting---protein expression

In vivo Animal

MTT assay

cancer cell lines stomach cancer SGC-7901 cells,BGC-803 cells[2]

human liver cancer BEL-7402 cells ， IC50 is 13. 78μM.[3, 4]

human liver cancer SMMC-7721 cells[2],HepG2 cells[5]（人肝癌细胞）

 Human breast cancer MCF-7 cells[2] [5]

human Rectum cancer LOVO cells[2](人直肠癌细胞 ) human Colon cancer LS-74T cells[2](人结肠癌细胞 ) human lung cancer A–549 cells[5, 6]（人肺癌细胞） Hela[5]cells(人宫颈癌细胞 ) CNE2[5](人鼻咽癌细胞 )

Hoechst 33342 staining

AO/EB staining

Coomassie brilliant blue staining(考马斯亮蓝染色 ) 正常对照组可清楚地看见细胞质内骨架蛋白呈蓝色丝状结构 ,核染色较致密，细胞呈不规则形。细胞骨架蛋白呈网格状结构，细胞间无重叠，相邻细胞之间可见骨架束彼此穿行

给药组细胞与周围细胞群脱离，表面原有的微绒毛、细胞间连接消失，有凋亡小体形成。Incubation with juglone resulted in obvious changes in the cell morphology. The formation of apoptotic bodies was observed

scanning electron microscopy(扫描电镜观察 ) 正常对照肿瘤组细胞呈多边形，细胞膜完整，表面微绒毛丰富、卷曲成丛状、规则排列、长丝突起分布均匀，细胞间壤嵌连接。

胡桃醌组胞体体积缩小，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。细胞间紧密连接结构消失，细胞表面长突起大部分消失，

细胞表面的微绒毛完全消失，胞体固缩，有部分细胞裂解，形成大小不一的凋亡小体状结构Incubation with juglone resulted in obvious changes in the cell morphology and cytoskeletal alterations of the cells.

Transmission electron microscopy,TEM(透射电镜 )

正常细胞形态规则，有丰富的微绒毛，核规则，以常染色体为主；胞浆丰富，电子密度低，线粒体嵴结构完整，并可见大量粗大内质网。

胡桃醌组可见凋亡细胞，体积明显变小，表面微绒毛消失，细胞膜完整，核膜皱褶，核内异染色质增多，染色质浓缩，边集于核膜，分叶状、花瓣状基底部紧贴核膜；内质网扩张，线粒体结构疏松，轻度肿胀，嵴消失。细胞表面微绒毛脱落。Scanning electron microscopy revealed reduced volume of the cell bodies, dissociation of the cells, curling and malformation of the microvilli on the cell surface with rupture of the intercellular junction and enlargement of the intercellular space.TEM showed nucleolar fragmentation and formation of the apoptotic bodies after the exposure to juglone for 24 h.

Flow cytometry (流式细胞仪 ) 12. 5μM 胡桃醌作用于 BEL-7402 后 , 细胞生长明显受限 , G0 / G1 期和 S 期细胞比例减少 , 而 G2 /M 期细胞增多 , 发生 G2 /M 期阻滞Cell cycle changes are analyzed by flow cytometry with cells at G1 phase significantly less than those of control and cells at G2 phase significantly more than those of control.

Western Blotting

Effect of Juglone on the protein level of Bcl-2 and Bax in HL-60 cells.A)Cells were treated with different concentrations of Juglone for 24h.B) B) Cells were treated with Juglone (1.5μg/mL) for different times.

Western Blotting

Effect of Juglone on procaspase-3, procaspase -9 and PARP proteins expression. A)Cells were treated with different concentrations of Juglone for 24h. B) Cells were treated with Juglone (1.5μg/mL) for different times.

Wang X. The expanding role of mitochondria in apoptosis. Genes Dev. 2001;15(22):2922-33

DiscussionAnti-cancer active

1. Juglone could inhibit the cell proliferation & induce apoptosis in a dose- and time-dependent manner in some cell lines.

Action mechanism

1. Juglone could induce apoptosis through mitochondrial pathway in some cell lines.

2. Juglone could increase the ROS and activate the mitochondrial pathway to induce apoptosis.

3. Juglone could activate ERK pathway and inhibit PI3K/AKT pathway.

Contradiction

P53In michelle’s study, Juglone can decrease P53

gene expression human fibroblasts.In Guan’s study, the expression of p53 have no

change in LS-174 cells.

Molecular mechanism of Cancer Target

Juglone