蟾皮中抗肿瘤小分子化合物的获取和抗肿瘤作用机制的研究
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摘要
第一部分蟾皮中抗肿瘤活性小分子化合物的获取和抗肿瘤作用机制研究
研究背景和目的
卫生部《2008中国卫生统计年鉴》中的资料显示,恶性肿瘤已经成为严重危害我国人民生命健康的首要因素,尤其在大城市中,其死亡率占所有死因构成比的28.84%,对我国人民的生活和健康形成了极大的威胁。从世界范围内来看,恶性肿瘤也已经成为人类的头号杀手。尽管恶性肿瘤可以通过手术、化疗、放疗等手段部分根治或缓解,抗肿瘤药物的研发在过去的十多年也取得了长足的进步,但手术治愈率低、放化疗毒副反应严重使患者难于承受的局面没有根本改观,继续寻找特异性强、高效低毒的抗肿瘤药物,仍然是医学领域的研究热点。
近年来,由于定向设计、合成先导药物及从组合化合物库中获得抗肿瘤药物前体愈来愈困难,并且化学药物相对毒副作用较强,所以人们已将目光转向天然药物。具有悠久历史并能有效治疗肿瘤的中药在医学界备受关注,经常作为放疗或者化疗的辅助方法。从中药中提取有效成分并将之开发成新药,是中药进入国际市场的一种有效方式。我国已在这一领域积累了一定的经验。从中药青蒿中研制出来的青蒿素及其衍生物的制剂,是目前治疗疟疾最好的药物,就是中药发展获得成功的一个典型。
已经从蟾蜍中分离出来的单体化合物有蟾毒灵、华蟾蜍精醇等蟾蜍二烯内酯类化合物,临床中多作为强心剂使用,对其机制的研究也较为透彻。现代中药学研究表明蟾皮及其制剂治疗肝癌、肺癌等恶性肿瘤疗效确切。目前在临床上广泛应用的蟾蜍来源的抗肿瘤制剂主要有华蟾素注射液、华蟾素胶囊等。但是因为工艺粗糙,纯度不高,成分复杂,易导致毒副反应,且抗肿瘤机理不明,故其治疗价值受限;此外,已经从蟾皮及其制剂中分离出的有抗肿瘤活性的化合物多为脂溶性,对于蟾皮中的水溶性组分尚未见有深入的开发。
本研究将采用生物检定导向的分离策略,借助于先进的分离纯化手段,对蟾皮水溶性组分进行了深入的分离纯化和活性研究,揭示蟾蜍中的药效组分对肿瘤细胞信号通路的调控作用,明确其抗肿瘤作用机理,为进一步开发天然抗肿瘤药物提供科学的理论依据及特异、有效的小分子药性化合物/先导化合物。
实验方法:
1.利用工业色谱、高效液相色谱和纯化工厂技术平台对蟾皮的组分进行层层深入的分离纯化;
2.采用CCK-8法对分离出的各级组分进行抗肿瘤活性鉴定;
3.应用流式细胞术分析蟾皮活性组分对肿瘤细胞周期的影响;
4.利用Western Blot和RT-PCR等分子生物学技术,初步探讨蟾皮活性组分调节肿瘤细胞中目的基因表达的情况;
5.建立活体成像荷瘤小鼠模型,长期监测药效组分体内治疗效果。
结果:
1.在蟾皮的4种工业色谱粗提组分中,Ⅳ号具有抗肿瘤活性;该组分在10-4mg/ml水平就表现出抑制肿瘤细胞增殖的效应,且随着浓度的升高而增强;
2.在Ⅳ号粗提组分进一步分离出的11种初筛组分中,第11号(HCS411)具有抗肿瘤活性,且活性强于IV号组分;
3.肝癌、肺癌、白血病和前列腺癌细胞对HCS411最敏感,乳腺癌、骨肉瘤和食管癌细胞较敏感,宫颈癌和结肠癌细胞不敏感;
4.与正常肝细胞相比较,HCS411对肝癌细胞呈现一定的特异性;
5. HCS411的抑制肿瘤细胞增殖活性较目前临床抗肿瘤常用的华蟾素注射液及顺铂均强;
6. HCS411可以通过诱导G2/M期阻滞抑制肿瘤细胞增殖;
7. HCS411抗肿瘤活性不依赖P53;
8. HCS411经纯化工厂分离后得到60个精细组分,有7个有较强的抗肿瘤活性;其中以2-SX048429活性最强;
9.荷瘤动物模型活体成像检测结果显示,2-SX048429活性成份在体内也有较强的抗肿瘤作用。
结论:
本研究采用生物鉴定导向的分离方法,首次从蟾皮可溶性组分中逐步分离出了具有抗肿瘤活性的7个精细组分,为进一步发现抗肿瘤小分子化合物奠定了良好的基础;对其抗肿瘤机制进行的初步研究表明,这些组分可以通过不依赖于p53的方式使肿瘤细胞阻滞于G2/M期,进而抑制肿瘤细胞增殖。
第二部分镍化合物通过调控细胞周期的致癌机制
研究背景和目的
肺癌是目前最常见的恶性肿瘤之一,也是全球发病率和死亡率增长最快的肿瘤。在世界大多数国家,肺癌的患病率和死亡率呈逐年上升的趋势,尤其是发达国家,肺癌在男性常见的恶性肿瘤中居首位。尽管目前对于肺癌的诊断和治疗方法较过去都有了很大改进,但肺癌的早期诊断困难、术后复发及转移率高等仍是临床研究的瓶颈问题。目前迫切需要从预防入手,从根源上控制这种对人类威胁极大的疾病。而要采取有效的预防措施,就必须深入了解肺癌发生的病因。虽然肿瘤的发生是环境因素和体内遗传因素相互作用的结果,但从总体上来看,环境因素是致癌的主要原因。据报导,80%~90%的人类癌症与环境中的理化因素有关,其中又以化学因素致癌为主。重金属镍及其化合物就是其中一种主要的环境致癌物。
流行病学资料显示,接触镍化合物与肺癌发病率的上升关系密切,但到目前为止,镍诱发肺癌的机制尚不清楚。研究者在多种肿瘤组织肺癌组织中都发现,cyclin B1呈过表达状态。近年的研究表明,镍化合物刺激调控的基因中的大多数的转录因子确定为由HIF-1α介导的低氧信号通路的靶蛋白。在肿瘤的发生和发展阶段,紊乱的细胞周期被认为是最重要的细胞内事件之一。在肿瘤发生中,基因突变的细胞被认为是必不可少的。在G1/S期转化过程中,cyclin D1和cyclin E是重要的调控因子,它们和许多肿瘤的发生相关,比如乳腺癌、肺癌和前列腺癌。此外,中国仓鼠卵巢细胞经镍处理后,G2/M期比例会明显增加,这通常和cyclin A和cyclin B相关。p53在G1/S期和G2/M期检查点起作用,其产物p53是一个转录因子,正常情况下很不稳定,这样就不能积累到足够高的水平而促进转录。所以,有必要研究清楚在镍致癌过程中所有的细胞周期蛋白的调控,包括cyclin D1、cyclin E、cyclin A和cyclin B,还有重要的细胞周期调控因子p53,以及它们在其中的生物学功能。
考虑到肺是镍化合物作用于体内的最主要的靶器官,本研究选择以人支气管上皮细胞Beas2B作为细胞模型,来研究镍化合物在肺癌发生中的机制,主要包括它对细胞周期中多种主要的周期蛋白的表达、细胞周期的进展以及细胞增殖的影响。
实验方法:
1.采用流式细胞术检测NiCl2刺激后Beas2B细胞的周期变化;
2.利用Western Blot和RT-PCR检测NiCl2处理的Beas2B细胞中cyclin D1、cyclin E、cyclin B1和cyclin A的表达水平;
3.应用报告基因检测的方法分析NiCl2对cyclin D1表达和P53活化的影响;
4.利用PI和cyclin A2双染法分析NiCl2处理前后Beas2B细胞M期的变化;
5.应用DNA重组技术构建pSuper/siCyclin B1表达质粒;
6.采用CCK-8法检测NiCl2对细胞增殖的影响。
结果:
1. NiCl2刺激的Beas2B细胞生长明显被抑制,流式细胞术检测发现细胞组滞于G2/M期;
2. NiCl2上调了Beas2B细胞中cyclin D1和cyclin E的表达;
3. HIF-1不参与NiCl2诱导的cyclin D1和cyclin E的表达;
4. P53不参与NiCl2诱导的G2/M期阻滞;
5. NiCl2刺激后cyclin B1的表达增加;
6.干扰cyclin B1后,NiCl2诱导的M期阻滞和生长抑制效应被显著削弱。
结论:
本研首次发现了在支气管上皮细胞中,镍化合物可以诱导cyclin D1和cyclin E表达上调并促进了细胞的G1/S期转换;转录因子HIF-1不参与这一过程。镍诱导的cyclin B1积累可以引起细胞的M期阻滞并最终导致细胞生长抑制;在细胞周期调控中发挥重要作用的蛋白p53没有参与其中。这些结果不仅加深了我们对于镍化合物致癌机制的理解,也为预防和治疗镍诱发的肿瘤作提供了新的思路。
Part I Isolation of Anticancer Compounds from Toad Skin and the Molecular Mechanism Study
Cancer has long been the number one killer in many parts of the world. Conventional cancer therapies such as surgery, chemotherapy and radiotherapy have a number of limitations such as relatively high cost, development of resistance and are often accompanied with many adverse side effects. Therefore, there is a pressing need to search for new drugs that are safe and effective for cancer treatments despite significant progress in oncology therapeutics in the last decades. Traditional Chinese medicines are sometimes used as an adjunct to radiotherapy or chemotherapy for cancer. Toad has been found to be a source of agents with anticancer properties for thousands of years in Chinese medicine. Actually, several compounds, such as bufalin and cinobufagin, had been successfully isolated from toad skin and always used in treating cardiovascular diseases. Clinical and laboratorial materials also showed that some of them had the effects of anticancer, but up to now, the exact constituents of toad skin that responsible for this inhibition of tumor have not been identified and its anticancer mechanism has not been fully understood.
In the present study, we fractionated spray-dried toad skin and used SMMC-7721 (human hepatoma cell line) and A549 (human lung adenocarcinoma epithelial cell line) to assess the inhibitory effects of the fractions. The proliferation of cells was detected by CCK-8 assay. The cell cycle distribution was analized by Flow Cytometry.
The spray-dried toad skin was fractionated by process-scale chromatography to give 4 fractions,Ⅰ,Ⅱ,ⅢandⅣ.CCK-8 assay demonstrated fractionⅣposses anti-tumor activity, while fractionsⅠ,ⅡandⅢnot. FractionⅣwere then fractionated by HPLC and 11 subfractions were yielded. Further cell proliferation assay showed that HCS411 has inhibitory effect on cancer cell growth and HCS408 has marginal effect, while other subfractions have not inhibitory effect. Further study demonstrated HCS411 significantly inhibited SMMC-7721 cell growth in a dose- and time-dependent manner. And we also found HCS411 triggered a notable G2/M cell cycle arrest in SMMC-7721 cells indicating that the growth inhibition of endothelial cells by HCS411 can be induced by the mitosis inhibition through the arresting at the G2/M phase of the cell cycle. Given the important role of p53 in cell cycle arrest, our result showed P53 was not involved in G2/M arrest in SMMC-7721 cells exposed to HCS411. Further HPLC analysis yielded 60 subfractions from HCS411, and 7 of which were verified to be effective to inhibit cancer cell growth. More importantly, we found that the subfraction 2-SX048429 was extremely potent to inhibit the xenograft tumor growth in vivo.
A strength of this study was our approach to active constituent identification, which was based on an unbiased, bioassaydirected fractionation of spray-dried toad skin, and yielded consistent and convincing results using relatively small amounts of material. The results of this study demonstrate that some subfractions in spray-dried toad skin may contain bioactive compounds with potential chemopreventive activity. These results lay a solid foundation for the generation of antitumor active microcompound from toad skin and are meanful to the development of new antitumor drug.
Part II Effects of Nickel on Cyclin Expression, Cell Cycle Progression and Cell Proliferation in Human Pulmonary Cells
The exposure to nickel has largely increased in industrial societies due to the environmental pollution by heavy metals at all stages of production, use, and disposal. Frequent exposure to high-concentrated nickel compounds has been considered as one of the potential causes of respiratory cancers. Previous studies have demonstrated that the cell signaling activation leading to cell cycle redistribution plays a critical role in carcinogenesis. In current study, notable increase of G1/S cell cycle transition was observed in human bronchial epithelial cells with 12 hours nickel exposure. The inductions of G1/S phase related cyclin D1 and cyclin E by nickel, which were demonstrated for the first time, may be responsible for the nickel induced G1/S transition. In addition, we verified that hypoxia-inducible factor-1alpha (HIF-1α), as an important transcription factor of nickel response, was not required for the cyclin D1 induction. On the other hand, Beas-2B cells treated with nickel compounds for 24 and 48 hours present obvious G2/M cell cycle arrest as well as significant cell proliferation inhibition. Given that the role of p53 in G2/M arrest has been widely proved, its contribution in nickel-induced G2/M arrest was excluded, respecting that its protein level, ser15 phosphorylation and transcriptional activity were not changed in nickel response. Further study revealed that cyclin B1, an important cyclin which prevents cells from M-phase exit and is frequently involved in the promotion of many kinds of tumor, was remarkably up-regulated by nickel on a HIF-independent manner, and the essential role of cyclin B1 in M-phase arrest and cell proliferation inhibition was further verified by knocking down its expression using specific small interference RNA. Taken together, our results demonstrate that nickel exposure can induce G1/S cell cycle transition by facilitating cyclin D1 and/or cyclin E expression, until the M-phase blockage caused by up-regulated cyclin B1 expression (after a latent phase) veiled the effect of cyclin D1 and/or cyclin E and lead to the overall cell cycle blockage and cell proliferation inhibition.
研究背景和目的
卫生部《2008中国卫生统计年鉴》中的资料显示,恶性肿瘤已经成为严重危害我国人民生命健康的首要因素,尤其在大城市中,其死亡率占所有死因构成比的28.84%,对我国人民的生活和健康形成了极大的威胁。从世界范围内来看,恶性肿瘤也已经成为人类的头号杀手。尽管恶性肿瘤可以通过手术、化疗、放疗等手段部分根治或缓解,抗肿瘤药物的研发在过去的十多年也取得了长足的进步,但手术治愈率低、放化疗毒副反应严重使患者难于承受的局面没有根本改观,继续寻找特异性强、高效低毒的抗肿瘤药物,仍然是医学领域的研究热点。
近年来,由于定向设计、合成先导药物及从组合化合物库中获得抗肿瘤药物前体愈来愈困难,并且化学药物相对毒副作用较强,所以人们已将目光转向天然药物。具有悠久历史并能有效治疗肿瘤的中药在医学界备受关注,经常作为放疗或者化疗的辅助方法。从中药中提取有效成分并将之开发成新药,是中药进入国际市场的一种有效方式。我国已在这一领域积累了一定的经验。从中药青蒿中研制出来的青蒿素及其衍生物的制剂,是目前治疗疟疾最好的药物,就是中药发展获得成功的一个典型。
已经从蟾蜍中分离出来的单体化合物有蟾毒灵、华蟾蜍精醇等蟾蜍二烯内酯类化合物,临床中多作为强心剂使用,对其机制的研究也较为透彻。现代中药学研究表明蟾皮及其制剂治疗肝癌、肺癌等恶性肿瘤疗效确切。目前在临床上广泛应用的蟾蜍来源的抗肿瘤制剂主要有华蟾素注射液、华蟾素胶囊等。但是因为工艺粗糙,纯度不高,成分复杂,易导致毒副反应,且抗肿瘤机理不明,故其治疗价值受限;此外,已经从蟾皮及其制剂中分离出的有抗肿瘤活性的化合物多为脂溶性,对于蟾皮中的水溶性组分尚未见有深入的开发。
本研究将采用生物检定导向的分离策略,借助于先进的分离纯化手段,对蟾皮水溶性组分进行了深入的分离纯化和活性研究,揭示蟾蜍中的药效组分对肿瘤细胞信号通路的调控作用,明确其抗肿瘤作用机理,为进一步开发天然抗肿瘤药物提供科学的理论依据及特异、有效的小分子药性化合物/先导化合物。
实验方法:
1.利用工业色谱、高效液相色谱和纯化工厂技术平台对蟾皮的组分进行层层深入的分离纯化;
2.采用CCK-8法对分离出的各级组分进行抗肿瘤活性鉴定;
3.应用流式细胞术分析蟾皮活性组分对肿瘤细胞周期的影响;
4.利用Western Blot和RT-PCR等分子生物学技术,初步探讨蟾皮活性组分调节肿瘤细胞中目的基因表达的情况;
5.建立活体成像荷瘤小鼠模型,长期监测药效组分体内治疗效果。
结果:
1.在蟾皮的4种工业色谱粗提组分中,Ⅳ号具有抗肿瘤活性;该组分在10-4mg/ml水平就表现出抑制肿瘤细胞增殖的效应,且随着浓度的升高而增强;
2.在Ⅳ号粗提组分进一步分离出的11种初筛组分中,第11号(HCS411)具有抗肿瘤活性,且活性强于IV号组分;
3.肝癌、肺癌、白血病和前列腺癌细胞对HCS411最敏感,乳腺癌、骨肉瘤和食管癌细胞较敏感,宫颈癌和结肠癌细胞不敏感;
4.与正常肝细胞相比较,HCS411对肝癌细胞呈现一定的特异性;
5. HCS411的抑制肿瘤细胞增殖活性较目前临床抗肿瘤常用的华蟾素注射液及顺铂均强;
6. HCS411可以通过诱导G2/M期阻滞抑制肿瘤细胞增殖;
7. HCS411抗肿瘤活性不依赖P53;
8. HCS411经纯化工厂分离后得到60个精细组分,有7个有较强的抗肿瘤活性;其中以2-SX048429活性最强;
9.荷瘤动物模型活体成像检测结果显示,2-SX048429活性成份在体内也有较强的抗肿瘤作用。
结论:
本研究采用生物鉴定导向的分离方法,首次从蟾皮可溶性组分中逐步分离出了具有抗肿瘤活性的7个精细组分,为进一步发现抗肿瘤小分子化合物奠定了良好的基础;对其抗肿瘤机制进行的初步研究表明,这些组分可以通过不依赖于p53的方式使肿瘤细胞阻滞于G2/M期,进而抑制肿瘤细胞增殖。
第二部分镍化合物通过调控细胞周期的致癌机制
研究背景和目的
肺癌是目前最常见的恶性肿瘤之一,也是全球发病率和死亡率增长最快的肿瘤。在世界大多数国家,肺癌的患病率和死亡率呈逐年上升的趋势,尤其是发达国家,肺癌在男性常见的恶性肿瘤中居首位。尽管目前对于肺癌的诊断和治疗方法较过去都有了很大改进,但肺癌的早期诊断困难、术后复发及转移率高等仍是临床研究的瓶颈问题。目前迫切需要从预防入手,从根源上控制这种对人类威胁极大的疾病。而要采取有效的预防措施,就必须深入了解肺癌发生的病因。虽然肿瘤的发生是环境因素和体内遗传因素相互作用的结果,但从总体上来看,环境因素是致癌的主要原因。据报导,80%~90%的人类癌症与环境中的理化因素有关,其中又以化学因素致癌为主。重金属镍及其化合物就是其中一种主要的环境致癌物。
流行病学资料显示,接触镍化合物与肺癌发病率的上升关系密切,但到目前为止,镍诱发肺癌的机制尚不清楚。研究者在多种肿瘤组织肺癌组织中都发现,cyclin B1呈过表达状态。近年的研究表明,镍化合物刺激调控的基因中的大多数的转录因子确定为由HIF-1α介导的低氧信号通路的靶蛋白。在肿瘤的发生和发展阶段,紊乱的细胞周期被认为是最重要的细胞内事件之一。在肿瘤发生中,基因突变的细胞被认为是必不可少的。在G1/S期转化过程中,cyclin D1和cyclin E是重要的调控因子,它们和许多肿瘤的发生相关,比如乳腺癌、肺癌和前列腺癌。此外,中国仓鼠卵巢细胞经镍处理后,G2/M期比例会明显增加,这通常和cyclin A和cyclin B相关。p53在G1/S期和G2/M期检查点起作用,其产物p53是一个转录因子,正常情况下很不稳定,这样就不能积累到足够高的水平而促进转录。所以,有必要研究清楚在镍致癌过程中所有的细胞周期蛋白的调控,包括cyclin D1、cyclin E、cyclin A和cyclin B,还有重要的细胞周期调控因子p53,以及它们在其中的生物学功能。
考虑到肺是镍化合物作用于体内的最主要的靶器官,本研究选择以人支气管上皮细胞Beas2B作为细胞模型,来研究镍化合物在肺癌发生中的机制,主要包括它对细胞周期中多种主要的周期蛋白的表达、细胞周期的进展以及细胞增殖的影响。
实验方法:
1.采用流式细胞术检测NiCl2刺激后Beas2B细胞的周期变化;
2.利用Western Blot和RT-PCR检测NiCl2处理的Beas2B细胞中cyclin D1、cyclin E、cyclin B1和cyclin A的表达水平;
3.应用报告基因检测的方法分析NiCl2对cyclin D1表达和P53活化的影响;
4.利用PI和cyclin A2双染法分析NiCl2处理前后Beas2B细胞M期的变化;
5.应用DNA重组技术构建pSuper/siCyclin B1表达质粒;
6.采用CCK-8法检测NiCl2对细胞增殖的影响。
结果:
1. NiCl2刺激的Beas2B细胞生长明显被抑制,流式细胞术检测发现细胞组滞于G2/M期;
2. NiCl2上调了Beas2B细胞中cyclin D1和cyclin E的表达;
3. HIF-1不参与NiCl2诱导的cyclin D1和cyclin E的表达;
4. P53不参与NiCl2诱导的G2/M期阻滞;
5. NiCl2刺激后cyclin B1的表达增加;
6.干扰cyclin B1后,NiCl2诱导的M期阻滞和生长抑制效应被显著削弱。
结论:
本研首次发现了在支气管上皮细胞中,镍化合物可以诱导cyclin D1和cyclin E表达上调并促进了细胞的G1/S期转换;转录因子HIF-1不参与这一过程。镍诱导的cyclin B1积累可以引起细胞的M期阻滞并最终导致细胞生长抑制;在细胞周期调控中发挥重要作用的蛋白p53没有参与其中。这些结果不仅加深了我们对于镍化合物致癌机制的理解,也为预防和治疗镍诱发的肿瘤作提供了新的思路。
Part I Isolation of Anticancer Compounds from Toad Skin and the Molecular Mechanism Study
Cancer has long been the number one killer in many parts of the world. Conventional cancer therapies such as surgery, chemotherapy and radiotherapy have a number of limitations such as relatively high cost, development of resistance and are often accompanied with many adverse side effects. Therefore, there is a pressing need to search for new drugs that are safe and effective for cancer treatments despite significant progress in oncology therapeutics in the last decades. Traditional Chinese medicines are sometimes used as an adjunct to radiotherapy or chemotherapy for cancer. Toad has been found to be a source of agents with anticancer properties for thousands of years in Chinese medicine. Actually, several compounds, such as bufalin and cinobufagin, had been successfully isolated from toad skin and always used in treating cardiovascular diseases. Clinical and laboratorial materials also showed that some of them had the effects of anticancer, but up to now, the exact constituents of toad skin that responsible for this inhibition of tumor have not been identified and its anticancer mechanism has not been fully understood.
In the present study, we fractionated spray-dried toad skin and used SMMC-7721 (human hepatoma cell line) and A549 (human lung adenocarcinoma epithelial cell line) to assess the inhibitory effects of the fractions. The proliferation of cells was detected by CCK-8 assay. The cell cycle distribution was analized by Flow Cytometry.
The spray-dried toad skin was fractionated by process-scale chromatography to give 4 fractions,Ⅰ,Ⅱ,ⅢandⅣ.CCK-8 assay demonstrated fractionⅣposses anti-tumor activity, while fractionsⅠ,ⅡandⅢnot. FractionⅣwere then fractionated by HPLC and 11 subfractions were yielded. Further cell proliferation assay showed that HCS411 has inhibitory effect on cancer cell growth and HCS408 has marginal effect, while other subfractions have not inhibitory effect. Further study demonstrated HCS411 significantly inhibited SMMC-7721 cell growth in a dose- and time-dependent manner. And we also found HCS411 triggered a notable G2/M cell cycle arrest in SMMC-7721 cells indicating that the growth inhibition of endothelial cells by HCS411 can be induced by the mitosis inhibition through the arresting at the G2/M phase of the cell cycle. Given the important role of p53 in cell cycle arrest, our result showed P53 was not involved in G2/M arrest in SMMC-7721 cells exposed to HCS411. Further HPLC analysis yielded 60 subfractions from HCS411, and 7 of which were verified to be effective to inhibit cancer cell growth. More importantly, we found that the subfraction 2-SX048429 was extremely potent to inhibit the xenograft tumor growth in vivo.
A strength of this study was our approach to active constituent identification, which was based on an unbiased, bioassaydirected fractionation of spray-dried toad skin, and yielded consistent and convincing results using relatively small amounts of material. The results of this study demonstrate that some subfractions in spray-dried toad skin may contain bioactive compounds with potential chemopreventive activity. These results lay a solid foundation for the generation of antitumor active microcompound from toad skin and are meanful to the development of new antitumor drug.
Part II Effects of Nickel on Cyclin Expression, Cell Cycle Progression and Cell Proliferation in Human Pulmonary Cells
The exposure to nickel has largely increased in industrial societies due to the environmental pollution by heavy metals at all stages of production, use, and disposal. Frequent exposure to high-concentrated nickel compounds has been considered as one of the potential causes of respiratory cancers. Previous studies have demonstrated that the cell signaling activation leading to cell cycle redistribution plays a critical role in carcinogenesis. In current study, notable increase of G1/S cell cycle transition was observed in human bronchial epithelial cells with 12 hours nickel exposure. The inductions of G1/S phase related cyclin D1 and cyclin E by nickel, which were demonstrated for the first time, may be responsible for the nickel induced G1/S transition. In addition, we verified that hypoxia-inducible factor-1alpha (HIF-1α), as an important transcription factor of nickel response, was not required for the cyclin D1 induction. On the other hand, Beas-2B cells treated with nickel compounds for 24 and 48 hours present obvious G2/M cell cycle arrest as well as significant cell proliferation inhibition. Given that the role of p53 in G2/M arrest has been widely proved, its contribution in nickel-induced G2/M arrest was excluded, respecting that its protein level, ser15 phosphorylation and transcriptional activity were not changed in nickel response. Further study revealed that cyclin B1, an important cyclin which prevents cells from M-phase exit and is frequently involved in the promotion of many kinds of tumor, was remarkably up-regulated by nickel on a HIF-independent manner, and the essential role of cyclin B1 in M-phase arrest and cell proliferation inhibition was further verified by knocking down its expression using specific small interference RNA. Taken together, our results demonstrate that nickel exposure can induce G1/S cell cycle transition by facilitating cyclin D1 and/or cyclin E expression, until the M-phase blockage caused by up-regulated cyclin B1 expression (after a latent phase) veiled the effect of cyclin D1 and/or cyclin E and lead to the overall cell cycle blockage and cell proliferation inhibition.
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