Andrographolide inhibits hepatoma cells growth and affects the expression of cell cycle related proteins

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1 药学学报 Acta Pharmaceutica Sinica 2009, 44 (9): Andrographolide inhibits hepatoma cells growth and affects the expression of cell cycle related proteins SHEN Kai-kai 1, LIU Tian-yu 1, XU Chong 1, JI Li-li 1, 2* 1, 2*, WANG Zheng-tao (1. Key Laboratory of Standardization of Chinese Medicines of Ministry of Education, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai , China; 2. Shanghai R&D Centre for Standardization of Chinese Medicines, Shanghai , China) Abstract: The present study is aimed to investigate the toxic effects of andrographolide (Andro) on hepatoma cells and elucidate its preliminary mechanisms. After cells were treated with different concentrations of Andro (0 50 μmol L 1 ) for 24 h, cell viability was evaluated with 3-(4,5-dimethylthiazol-2-yl) 2,5- diphenyltetrazolium bromide (MTT) assay. Furthermore, after hepatoma cells (Hep3B and HepG2) were treated with different concentrations of Andro (0 30 μmol L 1 ) for 14 d, the number of colony formation was accounted under microscope. Cell cycle related proteins such as Cdc-2, phosphorylated-cdc-2, Cyclin B and Cyclin D1 were detected with Western blotting assay and the cell cycle was analyzed by flow cytometry using propidium iodide staining. MTT results showed that Andro induced growth inhibition of hepatoma cells in a concentration-dependent manner but had no significant effects on human normal liver L-02 cells. Andro dramatically decreased the colony formation of hepatoma cells in the concentration-dependent manner. Moreover, Andro induced a decrease of Hep3B cells at the G 0 -G 1 phase and a concomitant accumulation of cells at G 2 -M phase. At the molecular level, Western blotting results showed that Andro decreased the expression of Cdc-2, phosphorylated-cdc-2, Cyclin D1 and Cyclin B proteins in a time-dependent manner, which are all cell cycle related proteins. Taken together, the results demonstrated that Andro specifically inhibited the growth of hepatoma cells and cellular cell cycle related proteins were possibly involved in this process. Key words: andrographolide; hepatoma cell; colony formation; cell cycle related protein CLC number: R285; R963 Document code: A Article ID: (2009) 穿心莲内酯抑制肝癌细胞生长及其对细胞内周期调控相关蛋白的影响 沈凯凯 1, 刘天瑜 1, 徐冲 1 1,, 季莉莉 2*, 王峥涛 1, 2* (1. 上海中医药大学中药研究所中药标准化教育部重点实验室, 上海 ; 2. 上海中药标准化研究中心, 上海 ) 摘要 : 本文主要探讨了穿心莲内酯在体外抑制肝癌细胞生长的作用及其机制 实验中采用 MTT 法检测穿心莲内酯 (0~50 μmol L 1 ) 对 L-02 Hep3B 和 HepG2 细胞存活率的影响, 通过软琼脂克隆形成实验检测穿心莲内酯 (0~30 μmol L 1 ) 与 Hep3B 和 HepG2 细胞孵育 14 d 后对其克隆形成率的影响, 通过流式细胞技术检测穿心莲内酯对 Hep3B 细胞周期的影响 同时采用蛋白印迹方法观察穿心莲内酯 (50 μmol L 1 ) 作用不同时间后对细胞周期相关蛋白如 Cdc-2 磷酸化 Cdc-2 Cyclin B 和 Cyclin D1 表达的影响 MTT 检测结果显示, 穿心莲内酯能明显抑制肝癌细胞株 (Hep3B 和 HepG2 细胞 ) 的生长并呈剂量依赖性关系, 而对人正常肝细胞 L-02 无明显作用 同时, 穿心莲内酯能够剂量依赖性地降低肝癌细胞株克隆形成率 流式细胞仪检测到穿心莲内酯 (50 μmol L 1 ) 作用 Hep3B 细胞后, G 0 -G 1 期细胞比例减少, G 2 -M 期细胞增多, 提示穿心莲内酯将细胞阻滞于 G 2 -M 期 蛋白印 Received Project supported by Shanghai Science and Technology Committee Grants (07ZR14107, 08JC ). * Corresponding author Tel: , Fax: , wangzht@hotmail.com; jll_syc@yahoo.com.cn

2 974 药学学报 Acta Pharmaceutica Sinica 2009, 44 (9): 迹实验发现, 穿心莲内酯 (50 μmol L 1 ) 作用 Hep3B 细胞不同时间后, 细胞内周期调控相关蛋白 Cdc-2 磷酸化 Cdc-2 Cyclin B 和 Cyclin D1 的表达下调 研究结果表明, 穿心莲内酯特异性地剂量依赖性地抑制肝癌细胞生长, 并诱导 Hep3B 细胞 G 2 -M 期阻滞, 其机制可能与下调 Cyclin D1 和 Cdc2/CyclinB 等周期相关蛋白有关 关键词 : 穿心莲内酯 ; 肝癌细胞 ; 克隆形成 ; 细胞周期相关蛋白 Hepatocellular carcinoma (HCC) is the most invasive and frequently diagnosed malignancy and is the fifth leading cause of cancer related deaths in the world, and the treatment outcomes of HCC are very poor [1, 2]. In recent years, seeking bioactive components isolated from traditional Chinese medicines (TCMs) applicable in treatment of tumor has attracted a great deal of interests all over the world. As we all know, the discoveries of taxol and camptothecin from plants are the important milestones in the history of anticancer drugs. Hence, it is possible to use bioactivity-directed components from TCMs that are efficacious in the treatment of HCC. Andrographis paniculata Nees (Acanthaceae), also known as King of Bitters, is widely used for the treatment of various diseases, including respiratory infection, inflammation, cold, fever, bacterial dysentery and diarrhea in China, India and other countries in subtropical and Southeast Asia [3 5]. Andrographolide (Andro, Figure 1), a natural diterpenoid lactone, is the major active component of A paniculata, which has been reported to possess a wide spectrum of biological activities, i.e. anti-inflammatory, antibiotic, immunomodulatory, anti-hiv, anti-tumor, anti-thrombotic and hepatoprotective effects [6 12]. In addition, recent pharmacological researches have demonstrated that Andro has potential anti-cancer activity against some malignant tumor cells such as HL-60, PC-3 and MCF-7 [13 15]. Now, the anti-tumor effects of Andro and its mechanisms have attracted a great deal of interests. Cell cycle is a series of events taken place in cell growth leading to its replication, which is regulated by various proteins including the cyclins, cyclin dependent kinases, and cyclin dependent kinase inhibitors [16]. In normal cells, this complex process is initiated only in the presence of a mitogenic stimulus. However, cancer cell progression lost the cell cycle checkpoints controls, as previous studies clarified; an up-regulated cell proliferation is a common characteristic in numerous cancers [17, 18]. Therefore, the cell cycle regulation signals for latent application in treatment for tumor have gradually aroused researcher's interest. In addition, many traditional herbs inhibit carcinoma cells growth via regulating cell cycle related proteins; however, it appears likely that different pathways are involved in different types of cells [19, 20]. The present study is designed to observe the anti-cancer effects of Andro and the possible involvement of cell cycle related proteins. Figure 1 Chemical structure of andrographolide (Andro) Materials and methods Chemicals, antibodies and reagents Andrographolide was isolated from leaves of Andrographis paniculata (Herbarium of Shanghai University of Traditional Chinese Medicine, Shanghai, China). Structure of Andro was elucidated based on 1 H NMR and 13 C NMR spectral evidences. The chemical structure is showed in Figure 1. The purity of the compound was more than 98% as determined with high pressure liquid chromatography (HPLC) analysis. Roswell Park Memorial Institute (RPMI) 1640 medium, Modified Eagle s medium (MEM), fetal bovine serum (FBS), penicillin and streptomycin were purchased from Gibco BRL (Gaithersburg, MD, USA). The following antibodies for Cdc-2, phosphorylated- Cdc-2 (Tyr15), Cyclin D1 and Cyclin B were from Cell Signaling Technology (Danvers, MA). The antibody for actin was from Santa Cruz Biotechnology (Santa Cruz, CA). Peroxidase-conjugated goat anti-rabbit IgG (H+L) and peroxidase-conjugated goat anti-mouse IgG (H+L) were from Jackson ImmunoResearch (West Grove, PA). Nitrocellulose membranes and prestained protein marker were from Bio-Rad (Hercules, CA) and enhanced chemiluminescence detection system was from Amersham Life Science (Buckinghamshire, UK).

3 SHEN Kai-kai, et al: Andrographolide inhibits hepatoma cells growth and affects the expression of cell cycle related proteins 975 All other reagents unless indicated were from Sigma Chemical Co (St. Louis, USA). Cell lines Human normal liver L-02 cells (Cell Bank, Type Culture Collection of Chinese Academy of Sciences, Shanghai) were cultured in RPMI 1640 supplemented with 10% (v/v) heat-inactivated FBS, penicillin (100 u ml 1 ) and streptomycin (100 µg ml 1 ), glutamine (2 mmol L 1 ) at 37 in a humidified atmosphere in the presence of 5% CO 2. Hepatomaderived Hep3B and HepG2 cells were obtained from the American Type Culture Collection (Manassas, VA, USA), and were maintained in MEM supplemented with 10% (v/v) heat-inactivated FBS, 2 mmol L 1 L-glutamine, penicillin (100 u ml 1 ), streptomycin (100 µg ml -1 ) and were incubated at 37 in a humidified, 5% CO 2 atmosphere. Cell viability assay Cells with density of were seeded in each well of 96-well microplates. After vehicle (0.1% DMSO) and andrographolide of various concentrations were added 24 h later, cells were incubated with 500 μg ml 1 3-(4,5-dimethylthiazol-2-yl) 2,5- diphenyltetrazolium bromide (MTT) for 4 h. The functional mitochondrial succinate dehydrogenases in survival cells can convert MTT to formazan that generates a blue color. At last the formazan was dissolved in 10% SDS 5% iso-butanol 0.01 mol L 1 HCl. The optical density was measured at 570 nm with 630 nm as a reference and cell viability was normalized as a percentage of control. Colony formation assay One thousand singlecell suspension of cells was resuspended in 1.5 ml growth media containing 0.3% low melting temperature agarose and were plated in 6-well plate over a base layer of 1.5 ml growth media containing 0.6% low melting temperature agarose. The plates were incubated for 14 d until colonies were formed. Colony cells more than 50 cells were counted as one positive colony under microscope. The inhibition of colony formation ratio was expressed in percentage of DMSO control. Flow cytometry fluorescence-activated cell sorting analysis Hep3B cells with density of were treated with Andro (50 μmol L 1 ) for indicated times and then collected and performed in a fluorescence-activated cell sorting (FACS) flow cytometer (BD sciences) with propidium iodide staining. The percentages of cell population at G 0 -G 1 and G 2 -M phases were observed. Western blotting analysis Following the treatments, cells were lysed in lysis buffer containing 50 mmol L 1 Tris (ph 7.5), 1 mmol L 1 EDTA, 150 mmol L 1 NaCl, 20 mmol L 1 NaF, 0.5% NP-40, 10% glycerol, 1 mmol L 1 phenylmethylsulfonyl fluoride, 10 μg ml 1 aprotinin, 10 μg ml 1 leupeptin and 10 μg ml 1 pepstatin A. Protein concentrations were assayed and normalized to equal protein concentration. Proteins were separated by SDS-PAGE and blots were probed with appropriate combination of primary and HRP conjugated secondary antibodies. For repeated immunoblotting, membranes were stripped in 62.5 mmol L 1 Tris (ph 6.7), 20% SDS and 0.1 mol L 1 2-mercaptoethanol for 30 min at 50. Statistical analysis For all experiments, data were expressed as x ± s. Statistical comparisons were subjected to an analysis of variance (ANOVA) and LSD-test using SPSS version 11.5, and P < 0.05 was considered significant. Results 1 Andro specifically induced cytotoxicity on hepatoma cells: Hep3B and HepG2 First, to test the hypothesis that hepatoma cells are more sensitive to Andro treatment, we initially investigated the cell growth inhibitory effects of Andro on human normal liver L-02 and hepatoma cells (Hep3B and HepG2). Cell survival curves for L-02, Hep3B and HepG2 cells after treatment with various concentrations of Andro ranging from 0 to 50 μmol L 1 for 24 h were showed in Figure 2. The results showed that under the treatment of Andro at 1, 5, and 10 μmol L 1, these three cell lines have little difference in cell viability. However, at higher concentrations of Andro ( 25 μmol L 1 ), there was significant difference among these cell lines. The cell viability of Hep3B cells had a dramatic decrease from 100.0% to 54.9%, and that of HepG2 cells decreased from 100.0% to 79.4%, while that of L-02 cells went down slightly only from to 100.0% to 94.6%. These results indicate that Andro can specifically inhibit the growth of hepatoma cells, especially Hep3B cells. 2 Andro decreased colony formation capability of hepatoma cells in soft agar media Next, we observed the effects of Andro on the colony formation capability in Hep3B cells. Cells were exposed to different concentrations of Andro ranging from 0 30 μmol L 1 for 14 days culture. As shown in

4 976 药学学报 Acta Pharmaceutica Sinica 2009, 44 (9): Figure 2 Andrographolide (Andro) inhibits cell growth of L-02, Hep3B and HepG2 cells, separately. Cells were seeded and treated with solvent (0.1% DMSO) or different concentrations of Andro (0 50 μmol L 1 ) for 24 h. The survival cells were determined with MTT (n = 5, x ± s). ### P < vs DMSO control group (HepG2 cells); *** P < vs DMSO control group (Hep3B cells) Figure 3A, the number of colony formation significantly decreased in Hep3B cells with incubation of high concentrations of Andro (30.0, 10.0, and 3.0 μmol L 1 ). The administration of 30.0, 10.0, 3.0, 1.0, and 0.3 μmol L 1 of Andro resulted in the inhibition of colony formation capability of Hep3B cells by (100.0 ± 0.0) %, (100.0 ± 0.0) %, (99.1 ± 1.7) %, (75.9 ± 2.5) % and (18.6 ± 9.5) %, respectively, which is in a concentrationdependent manner. Further, we observed the effects of Andro on the colony formation capability in HepG2 cells. The results showed that Andro could also decrease colony formation capability in a concentrationdependent manner (Figure 3B), which further confirmed the inhibitory effects of Andro on hepatoma cells. 3 Andro caused cell growth arrest at G 2 -M phase and regulated the expression of cell cycle related proteins Previous studies have shown that cell cycle proteins are up-regulated in many cancer cells [17, 18] and are associated with proliferative and anti-apoptotic pathways. In the present study we postulates that the anti-carcinogenic activity of Andro may be due, in part, to down-regulate of cell cycle proteins. To examine the mechanism responsible for Andro-mediated cell growth inhibition, cell cycle distribution was evaluated using FACS. The results shown in Figure 4 revealed Figure 3 Andrographolide (Andro) inhibited colony formation capability of hepatoma cells in soft agar media. Decreased colony formation units in Hep3B (A) and HepG2 (B) cells. One thousand single-cell suspension of cells treated with solvent (DMSO) or different concentrations of Andro ( μmol L 1 ) were moved to 0.3% low melting temperature agarose and overlaid on solidified 0.6% low melting temperature agarose in a 6-well plate for 14 days incubations. The inhibition of colony formation ratio is expressed as the percentage of the plating efficiency of Andro-treated cells compared to DMSO control (n = 5, x ± s). a: Andro (0 μmol L 1 ); b: Andro (0.3 μmol L 1 ); c: Andro (1.0 μmol L 1 ); d: Andro (3.0 μmol L 1 ); e: Andro (10.0 μmol L 1 ); f: Andro (30.0 μmol L 1 ). *** P < vs DMSO control group

5 SHEN Kai-kai, et al: Andrographolide inhibits hepatoma cells growth and affects the expression of cell cycle related proteins 977 Figure 4 Andro induced cycle arrest in Hep3B cells. Cells were treated with Andro (50 μmol L 1 ) for indicated times and then trypsinized, fixed, and stained with PI to measure cell cycle by FACS. n = 2, x ± s. ** P < 0.01 vs control group that Andro induced a decrease of Hep3B cells at the G 0 -G 1 phase, from 56.63% to 50.41%, and a concomitant accumulation of cells at G 2 -M phase, from 10.53% to 16.91%. Further, we detected the expression of Cyclin B and Cdc-2 using Western blotting. The results in Figure 5 revealed that treatment of Hep3B cells with Andro (50 μmol L 1 ) for various times (0 16 h) led to a marked decline in protein levels of Cyclin B and Cdc-2 with a time-dependent manner. The results further Figure 5 Andro decreased cellular expression of Cdc-2, phosphorylated-cdc-2, Cyclin D1 and Cyclin B in Hep3B cells. Hep3B cells were treated with DMSO or Andro (50 μmol L 1 ) for various hours (0 16 h), and expression of proteins in whole-cell lysates was determined with Western blotting analysis. The result represents at least two repeats suggest that the potent growth-inhibitory activity of Andro on Hep3B cells may be due to its interference with the cell cycle process. Discussion Andro is a bioactive diterpenoid lactone from A paniculata. In this study, our results showed that Andro specially inhibited the cell growth in hepatoma cell lines (Hep3B and HepG2) (Figure 2), which indicates that Andro can be potentially applied in the treatment of HCC. Previous reports demonstrated that URE (methanolic extract of Uncaria rhynchophylla) had high cytotoxic effects on HT-29 cells by using colony formation assay, and indicated that it was a potential chemotherapeutic agent candidate against HT-29 human colon carcinoma cells [21]. Also, it has been reported that natural and synthetic vitamin E had anticancer actions on human MDA-MB-435 and MCF-7 breast cancer cells via inhibition of colony formation [22]. Characteristics of carcinoma cells are capable of forming colonies in soft agar media as previous studies demonstrated. The results in this study (Figure 3A and 3B)

6 978 药学学报 Acta Pharmaceutica Sinica 2009, 44 (9): showed that Andro could decrease colony formation capability in a concentration-dependent manner in hepatoma cells, which further confirm that Andro has potential effect on the treatment of cancer. Further FACS analysis (Figure 4) demonstrated that Andro induced the accumulation of cells at G 2 -M phase, which indicates that the growth-inhibitory effect of Andro on Hep3B cells may be partly due to inducing G 2 -M cell cycle arrest. The complex formed by the association of Cyclin B and cell division cycle protein 2 (Cdc-2) is a key intracellular regulator in the entry into mitosis [23]. Western blotting analysis (Figure 5) demonstrated that Andro triggered significant reduction at the levels of Cdc-2 kinase and Cyclin B, which may contribute to the arrest of G 2 -M. Cyclin D1 is a key regulator involved at G 0 -G 1 checkpoint. Our results showed that Andro reduced the level of Cyclin D1 protein. Previous study has showed that Andro inhibited NF-κB activation, as Cyclin D1 is a downstream target of NF-κB, which maybe the reason leading to its decline [24, 25]. In general, the enhanced expression of phosphorylated-cdc-2 is the common characteristic of cell cycle arrest at G 2 -M phase. However, we found that Andro treatment decreased the level of phosphorylated-cdc-2, which may be, in part, due to the depletion of Cdc-2 protein. Taken together, Andro treatment might cause a reduction in kinase activity of the Cdc-2 / Cyclin B complex to elicit G 2 -M phase cell cycle arrest mainly because of the decline in the protein levels of Cyclin B and Cdc-2. Previous study has reported that the anti-cancer activity of Andro and DRF3188 (a novel semi-synthetic analog of Andro) on MCF-7 cells mainly through inducing p27 expression and concomitant decrease of Cdk4 levels to block the G 0 -G 1 phase of the cell cycle [15]. Similarly, Andro inhibited cell-cycle progression in Lovo cells by decreasing the protein expression of Cyclin A, Cyclin D1, Cdk2 and Cdk4 [10]. In the present study, our results demonstrated that Andro specifically inhibited the growth of HCC cell line Hep3B and HepG2 possibly via down-regulating cell cycle related proteins and arresting cell growth at G 2 -M phase. Taken together, the present study provides pharmacological support that Andro may act as a novel promising agent for clinical applications in the treatment of HCC. However, the exact mechanisms of the anti-hcc effects of Andro and its therapeutic efficacy in vivo should be further investigated. References [1] Parkin DM, Bray F, Ferlay J, et al. Global cancer statistics, 2002 [J]. CA Cancer J Clin, 2005, 55: [2] Colombo M. Natural history of hepatocellular carcinoma [J]. Cancer Imaging, 2005, 5: [3] Poolsup N, Suthisisang C, Prathanturaruq S, et al. Andrographis paniculata in the symptomatic treatment of uncomplicated upper respiratory tract infection: systematic review of randomized controlled trials [J]. J Clin Pharm Ther, 2004, 29: [4] Deng WL. Outline of current clinical and pharmacological research on Andrographis paniculata in China [J]. Newsl Chin Herb Med, 1978, 10: [5] Coon JT, Ernst E. Andrographis paniculata in the treatment of upper respiratory tract infections: a systematic review of safety and efficacy [J]. Planta Med, 2004, 70: [6] Abu-Ghefreh AA, Canatan H, Ezeamuzie CI. In vitro and in vivo anti-inflammatory effects of andrographolide [J]. Int Immunopharmacol, 2009, 9: [7] Wu CM, Cao JL, Zheng MH, et al. Effect and mechanism of andrographolide on the recovery of Pseudomonas aeruginosa susceptibility to several antibiotics [J]. J Int Med Res, 2008, 36: [8] Carretta MD, Alarcon P, Jara E, et al. Andrographolide reduces IL-2 production in T-cells by interfering with NFAT and MAPK activation [J]. Eur J Pharmacol, 2009, 602: [9] Basak A, Cooper S, Roberqe AG, et al. Inhibition of proprotein convertases-1,-7 and furin by diterpines of Andrographis paniculata and their succinoyl esters [J]. Biochem J, 1999, 338: [10] Shi MD, Lin HH, Lee YC, et al. Inhibition of cell-cycle progression in human colorectal carcinoma Lovo cells by andrographolide [J]. Chem Biol Interact, 2008, 174: [11] Li YD, Ye BQ, Zheng SX, et al. NF-kappa B transcription factor p50 critically regulates tissue factor in deep vein thrombosis [J]. J Biol Chem, 2009, 284: [12] Trivedi NP, Rawal UM, Patel BP. Hepatoprotective effect of andrographolide against hexachlorocyclohexane-induced oxidative injury [J]. Integr Cancer Ther, 2007, 6: [13] Manikam ST, Stanslas J. Andrographolide inhibits growth of acute promyelocytic leukaemia cells by inducing retinoic acid receptor-independent cell differentiation and apoptosis [J]. J Pharm Pharmacol, 2009, 61: [14] Kim TG, Hwi KK, Hunq CS. Morphological and biochemical

7 SHEN Kai-kai, et al: Andrographolide inhibits hepatoma cells growth and affects the expression of cell cycle related proteins 979 changes of andrographolide-induced cell death in human prostatic adenocarcinoma PC-3 cells [J]. In Vivo, 2005, 19: [15] Satyanarayana C, Deevi DS, Rajagopalan R, et al. DRF 3188 a novel semi-synthetic analog of andrographolide: cellular response to MCF 7 breast cancer cells [J]. BMC Cancer, 2004, 4: [16] Norbury C, Nurse P. Animal cell cycles and their control [J]. Annu Rev Biochem, 1992, 61: [17] MacLachian TK, Sang N, Giordano A. Cyclins, cyclindependent kinases and cdk inhibitors: implications in cell cycle control and cancer [J]. Crit Rev Eukaryot Gene Expr, 1995, 5: [18] Spataro V. Recent advances in the molecular genetics of cancer [J]. Ann Oncol, 1998, 9: [19] Chiu LC, Wan JM. Induction of apoptosis in HL-60 cells by Lett, 2000, 160: [21] Jo KJ, Cha MR, Lee MR, et al. Methanolic extracts of Uncaria rhynchophylla induce cytotoxicity and apoptosis in HT-29 human colon carcinoma cells [J]. Plant Foods Hum Nutr, 2008, 63: [22] Yu WP, Jia L, Wang P, et al. In vitro and in vivo evaluation of anticancer actions of natural and synthetic vitamin E forms [J]. Mol Nutr Food Res, 2008, 52: [23] Sancar A, Lindsev-Boltz LA, Unsal-Kacmaz K, et al. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints [J]. Annu Rev Biochem, 2004, 73: [24] Iruretaqovena MI, Sepulveda SE, Lezana JP, et al. Inhibition of nuclear factor-kappa B enhances the capacity of immature dendritic cells to induce antigen-specific tolerance in experimental autoimmune encephalomyelitis [J]. Ther, 2006, 318: J Pharmacol Exp eicosapentaenoic acid (EPA) is associated with downregulation of bcl-2 expression [J]. Cancer Lett, 1999, 145: [25] Bao Z, Guan S, Chenq C, et al. A novel anti-inflammatory role for andrographolide in asthma via inhibition of the nuclear [20] Chan FL, Choi HL, Chen ZY, et al. Induction of apoptosis in prostate cancer cell lines by a flavonoid, baicalin [J]. Cancer factor-kappab pathway [J]. 2009, 179: Am J Respir Crit Care Med, 热分析在药物及药用材料中的应用学术研讨会通知 ( 第二轮 ) 随着现代科学仪器的发展, 热分析在药物及药用材料领域中的应用越来越广泛, 中国药典 日本药局方 美国药典 英国药典 2009 年版均收载了热分析方法 在化学药物 生化药物 抗生素 中药化学 药物包装材料 药物质量评价 新药研究中处方筛选及有关制剂的质量分析等方面, 热分析法都起着非常重要的作用 为此, 药物分析杂志编委会定于 2009 年 10 月 22~24 日在苏州召开全国药物分析及药用材料热分析法学术研讨会 此次会议由江苏省食品药品检验所承办, 江苏省热分析专业委员会协办, 会上拟请有关专家作专题报告, 并开展有关专题讨论 有关会议详情及参会事宜请登陆网站 : 联系人 : 刘小帅, 于宝珠 ( ) 电话 : (010) ; 传真 : (010) ; 地址 : 北京天坛西里 2 号中国药品生物制品检定所内药物分析杂志编辑部 ( 邮编 )