新型蛋白酶体抑制剂地钱素M诱导前列腺癌PC3细胞凋亡的研究
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摘要
前列腺癌(prostate cancer, PCa)作为常见的男性恶性肿瘤,在我国的发病率较欧美国家低,但近年来呈明显的增长趋势,且中晚期患者的比例远远大于国外,由于PCa潜伏期长,发病机制复杂,尚无有效的治疗手段,因此,PCa的预防与治疗一直备受关注。目前激素撤除治疗是中晚期前列腺癌的首选方法,通过手术、抗雄激素类药物的激素阻断治疗,对约80%的前列腺癌有效。然而绝大多数肿瘤经治疗后,PCa发展为激素难治性前列腺癌(hormone refractory prostate cancer, HRPC),对抗内分泌治疗,预后差,死亡率高。PCa由激素治疗敏感到耐受的演变是复杂的多因素、多信号途径参与的过程。
尽管对HRPC的病理机制有相当深入的研究,至今临床无有效的治疗方案,化疗依然是主要的治疗手段。针对雄激素受体途径、抗凋亡基因、血管生成因子、生长因子受体、信号通路蛋白等靶点的各种化学药物、疫苗、单克隆抗体等均能有效抑制前列腺癌细胞增殖,诱导其凋亡,显示出良好的效果。如多西紫杉醇常用于晚期HRPC的治疗,通过稳定微管、诱导凋亡而发挥作用,是唯一被证实可明显延长HRPC患者生存期的药物,使HRPC的化疗取得了新的进展,同时也显示出天然产物作为抗肿瘤药物的巨大潜力。因此,研究HRPC的发病机制、探索新的治疗靶点及方法,是预防与治疗HRPC的有效策略。
泛素-蛋白酶体(ubiquitin-proteasome system, UPS)是哺乳动物细胞内主要的蛋白水解酶体系,能够精确降解细胞内各种靶蛋白,对于细胞周期运转、细胞分化与凋亡、免疫应答、信号转导等生理过程起着重要的调控作用。对恶性肿瘤细胞而言,蛋白代谢紊乱,UPS调节失衡,使UPS与肿瘤的发生发展、多药耐药、免疫监控逃逸等病理过程密切相关。而抑制UPS对肿瘤细胞比正常细胞更为敏感,可导致细胞周期阻滞、细胞凋亡等。因此UPS成为肿瘤治疗的新靶点,蛋白酶体抑制剂(proteasome inhibitors)也成为一种非常有前景的抗肿瘤药物。因此,发现新的蛋白酶体抑制剂为抗肿瘤药物的研究提供了新思路,而天然产物以其独特的结构类型更是受到研究者的青睐。
苔藓植物作为从水生到陆生过渡的一类高等植物,品种繁多,抗虫害、抗腐烂等独特的生物特性导致其化学成分结构多样,包括萜类化合物、酚类化合物和联苄类化合物等,是发现药效活性分子的宝贵资源。双联苄类化合物作为苔藓植物的成分之一,是新型植物抗生素,具有广泛的生理活性,具有抗菌、抗病毒、细胞毒等活性。我们通过对50多种联苄类化合物进行抗肿瘤活性筛选,发现其中一些化合物具有很好的抑制肿瘤细胞增殖活性。双联苄化合物地钱素M(Marchantin M, Mar)具有抑制肿瘤细胞增殖活性,且对前列腺肿瘤细胞的抑瘤活性显著。本文就双联苄化合物地钱素M抑制激素非依赖性前列腺癌PC3细胞增殖的机制进行了初步探索。
首先通过细胞增殖实验检测地钱素M的抑瘤活性,结果显示地钱素M对人肝癌HepG2细胞的抑制作用较弱,对人白血病K562细胞和腺体瘤包括人乳腺癌MCF-7细胞、前列腺癌LNCaP、DU145和PC3细胞抑制作用比较明显,特别是对激素非依赖性前列腺癌PC3细胞的增殖抑制作用尤为显著,而地钱素M对正常人视网膜上皮细胞hTERT-RPE1细胞毒性作用较小,表明肿瘤细胞对地钱素M更为敏感。
BrdU掺入实验表明,地钱素M以浓度依赖的方式显著抑制PC3细胞的增殖活性。流式细胞术分析结果显示,地钱素M阻滞PC3细胞于G0／G1期。分析G0／G1期周期相关蛋白的表达,结果显示,地钱素M诱导周期蛋白激酶抑制蛋白p21~(cip1)的表达,抑制CyclinDl的表达,同时使Rb蛋白的磷酸化程度降低,抑制细胞周期G1／S期的转变。另外,流式细胞术分析结果出现显著的亚二倍体峰,提示地钱素M抑制细胞增殖可能与诱导细胞凋亡有关。进一步研究发现,经地钱素M处理后的PC3细胞的形态发生显著变化,Giemsa染色和PI/Hoechest33342双染结果显示,细胞变圆,脱落明显,且细胞形态出现凋亡细胞的特征性改变,DAPI染色观察到细胞核发生明显的凋亡性变化。Westernblotting结果显示,地钱素M处理后,PC3细胞内的抗凋亡蛋白Bcl2的表达下调,而促凋亡蛋白Bax的表达明显增高,其Bax/Bcl2的比率显著增加。由于Caspase在细胞凋亡中发挥重要作用,我们的检测结果表明,地钱素M能够激活PC3细胞中Caspase 3活性,促进多聚ADP-核糖聚合酶(PARP)的剪切,说明地钱素M可能以Caspase依赖的方式诱导PC3细胞凋亡。而经地钱素M处理后,细胞内Caspase 9和Caspase 4的活性增加,但Caspase 8活性并无明显改变,表明线粒体、内质网途径可能参与地钱素M诱导PC3细胞凋亡的作用,但其机制仍需进一步研究。由于地钱素M与茶多酚的结构相似,后者已被证实具有抑制蛋白酶体的作用,因而我们检测地钱素M对蛋白酶体的酶解活性的影响。检测结果显示,地钱素M在体外显著抑制纯化的20S蛋白酶体的糜蛋白酶样活性、多肽／谷氨酰水解酶活性,其抑制作用随浓度的增加而增强。而经地钱素M作用后PC3细胞内糜蛋白酶样活性、多肽／谷氨酰水解酶活性随着地钱素M作用浓度的增加、作用时间的延长而显著降低,表明地钱素M具有明显的抑制蛋白酶体活性的作用。因此,地钱素M可能是通过抑制蛋白酶体活性发挥了诱导PC3细胞凋亡的作用。
总之,本研究结果表明,双联苄类化合物地钱素M具有抑制激素非依赖性前列腺癌PC3细胞增殖、引起细胞周期阻滞、诱导细胞凋亡的作用。其作用机制可能是通过抑制蛋白酶体活性诱导细胞凋亡,线粒体和内质网可能参与其诱导凋亡的过程。本研究首次报道一种新型的蛋白酶体抑制剂,为天然的双联苄化合物的进一步开发利用提供了新的思路。
Prostate cancer (prostate cancer, PCa) is the most common malignant cancer and the second cause of death in men in Europe and America countries. Recently, prostate cancer has become more common in our country.
PCa is an androgen-dependent tumor, and androgen ablation serves as an standard treatment for patients who present advanced, androgen-dependent metastatic PCa for many years. However, the high failure rate for castration causes progresses to hormone refractory prostate cancer (HRPC), that is resistant to radiation, surgery, and chemotherapy. It is the major cause of the mortality associated with HRPC. Despite numerous advances including aberrant regulation of androgen/androgen receptor leading to constitutive active androgen receptor, activation of MAPK, PI3K/Akt, JAK-STAT signaling pathways which contribute cell proliferation in addition to androgen stimuli in PCa, abnormal control of apoptosis cascade such as alterations of the expression of Bcl2, Bax resulting in apoptosis resistance, the high mortality rate occurs because the treatment of this stage of PCa has been largely ineffective, eventually the patients would die of an HRPC.
To date, chemotherapy is still a common clinical strategy for HRPC. For example, the pacitaxel, Present clinical drugs has their own chemical and biological characteristics, yet treatment outcomes are not satisfactory. The mechanism includes two aspects. (1)Patients with advanced prostate cancers undergo distant metastasis, and have many complications. (2) Despite rapid symptoms improved and lifetime prolonged, serious side effects of chemical drugs cause damage of body, repress immune system and result in poor prognosis. For example, Chepatotoxicity though with the advantages of wide spectrum anti-tumor effects and strong effects; Cyclophosphamide exhibits bone marrow depression; docetaxel triggers cancer cells to produce drug resistance. Therefore, it is required urgently to find novel drugs with high effects and low toxicity to treat the androgen-independent PCas.
Ubiquitin-proteasome system (UPS), the major proteolytic enzyme system in mammalian cells, plays critical roles in basic cellular functional process, including proliferation, differentiation and proliferation, and so on. As a proteolytic enzyme complex, UPS is an ATP dependent proteolytic complex, which is comprised of 20 S catalytic particle, 11S regulatory factor and two 19S regulatory particles. Maintaining the activity of UPS is greatly involved in keeping the maintenance of cell basic functions. UPS precisely degrades target proteins in mammalian cells, and then participates in the regulation of gene transcription and cell cycle, as well as in the cellular physical process which includes receptor-mediated endocytosis and antigen processing. Therefore, UPS will be an important target for drug treatment. Recent studies have validated that malignant tumors are more sensitive to UPS inhibitors compared to non-malignant tumors. Our study found that Marchantin M could inhibit the activity of UPS and the proliferation of PC3 cells.
At present, natural compound with anticancer constituents is highly concerned. liverworts contains a number of natural compound, including terpenoid, phenols and bisbibenzyls, among which bisbibenzyls with unique chemical structure and wide biological property receive great attention. We screened many bisbibenzyls candidates and found that Marchantin M exhibited inhibiting effect of proteasome activity and had significantly anticancer activity of PCa PC3 cells. The mechanism by which Marchantin M inhibited proliferation of PC3 cells and activity of proteasome was discussed in our study.
Cell proliferation assay, a method to test antitumor activity of Marchantin M, showed that Marchantin M remarkably repressed proliferation of adenocarcinoma cells including breast cancer MCF-7 cells, prostate cancer LNCaP, DU145 and PC3 cells. This effect was especially evident in androgen-independent PCa PC3 cells. Additionally, Marchantin M was of low toxicity in normal human retinal epithelium cells. Marchantin M significantly depressed PC3 cells in the concentration of 20μmol/L. Yet, in this concentration, it hardly had repression in hTERT-RPE1. Cell morphological alterations were observed by Gimsa staining and double staining (PI and Hoechest33342) methods. The results indicated that cells remarkably dropped off and cell morphology exhibited the characteristic changes of apoptosis. Next, we tested cell cycle by flow cytometry and western blotting, and found that apoptosis rate increased dramatically compared to the control group and cells were apparently arrested in G0/G1 phase. Changes of apoptosis related proteins by western bolting exhibited that anti-apoptosis protein Bcl2 was down-regulated whereas pro-apoptosis was significantly increased in treatment group. In the concentration of 10μmol/L, we found activation of Caspase 3, cleavage of PARP, and activation of Caspase 4 and Caspase 9. However, obvious activation of Caspase 8 was not observed. These data indicated that Marchantin M inhibited proliferation of PC3 cells by inducing apoptosis of them. Further study was made on the mechanism of apoptosis in PC3 cells by Marchantin M. Structure of Marchantin M has been validated to be similar to a proteasome inhibitor (tea polyphenol) in recent studies. So we supposed that Marchantin M might induce apoptosis of PC3 cells by inhibiting activity of proteasome. we detected activity of proteasome and found Marchantin M could inhibit enzymolysis activity of proteasome in vitro and also repress the enzymolysis activity of proteasome for total proteins in PC3 cells. This was consistent with our hypothesis.
In summary, our study showed that Marchantin M could significantly inhibit proliferation of androgen-independent PCa PC3 cells and increase apoptosis of PC3 cells. The potential mechanism is that repression of UPS lead to cell apoptosis. In our study, the novel proteasome inhibitor to cope with PCa will give new hope for treatment of human PCa.
尽管对HRPC的病理机制有相当深入的研究,至今临床无有效的治疗方案,化疗依然是主要的治疗手段。针对雄激素受体途径、抗凋亡基因、血管生成因子、生长因子受体、信号通路蛋白等靶点的各种化学药物、疫苗、单克隆抗体等均能有效抑制前列腺癌细胞增殖,诱导其凋亡,显示出良好的效果。如多西紫杉醇常用于晚期HRPC的治疗,通过稳定微管、诱导凋亡而发挥作用,是唯一被证实可明显延长HRPC患者生存期的药物,使HRPC的化疗取得了新的进展,同时也显示出天然产物作为抗肿瘤药物的巨大潜力。因此,研究HRPC的发病机制、探索新的治疗靶点及方法,是预防与治疗HRPC的有效策略。
泛素-蛋白酶体(ubiquitin-proteasome system, UPS)是哺乳动物细胞内主要的蛋白水解酶体系,能够精确降解细胞内各种靶蛋白,对于细胞周期运转、细胞分化与凋亡、免疫应答、信号转导等生理过程起着重要的调控作用。对恶性肿瘤细胞而言,蛋白代谢紊乱,UPS调节失衡,使UPS与肿瘤的发生发展、多药耐药、免疫监控逃逸等病理过程密切相关。而抑制UPS对肿瘤细胞比正常细胞更为敏感,可导致细胞周期阻滞、细胞凋亡等。因此UPS成为肿瘤治疗的新靶点,蛋白酶体抑制剂(proteasome inhibitors)也成为一种非常有前景的抗肿瘤药物。因此,发现新的蛋白酶体抑制剂为抗肿瘤药物的研究提供了新思路,而天然产物以其独特的结构类型更是受到研究者的青睐。
苔藓植物作为从水生到陆生过渡的一类高等植物,品种繁多,抗虫害、抗腐烂等独特的生物特性导致其化学成分结构多样,包括萜类化合物、酚类化合物和联苄类化合物等,是发现药效活性分子的宝贵资源。双联苄类化合物作为苔藓植物的成分之一,是新型植物抗生素,具有广泛的生理活性,具有抗菌、抗病毒、细胞毒等活性。我们通过对50多种联苄类化合物进行抗肿瘤活性筛选,发现其中一些化合物具有很好的抑制肿瘤细胞增殖活性。双联苄化合物地钱素M(Marchantin M, Mar)具有抑制肿瘤细胞增殖活性,且对前列腺肿瘤细胞的抑瘤活性显著。本文就双联苄化合物地钱素M抑制激素非依赖性前列腺癌PC3细胞增殖的机制进行了初步探索。
首先通过细胞增殖实验检测地钱素M的抑瘤活性,结果显示地钱素M对人肝癌HepG2细胞的抑制作用较弱,对人白血病K562细胞和腺体瘤包括人乳腺癌MCF-7细胞、前列腺癌LNCaP、DU145和PC3细胞抑制作用比较明显,特别是对激素非依赖性前列腺癌PC3细胞的增殖抑制作用尤为显著,而地钱素M对正常人视网膜上皮细胞hTERT-RPE1细胞毒性作用较小,表明肿瘤细胞对地钱素M更为敏感。
BrdU掺入实验表明,地钱素M以浓度依赖的方式显著抑制PC3细胞的增殖活性。流式细胞术分析结果显示,地钱素M阻滞PC3细胞于G0／G1期。分析G0／G1期周期相关蛋白的表达,结果显示,地钱素M诱导周期蛋白激酶抑制蛋白p21~(cip1)的表达,抑制CyclinDl的表达,同时使Rb蛋白的磷酸化程度降低,抑制细胞周期G1／S期的转变。另外,流式细胞术分析结果出现显著的亚二倍体峰,提示地钱素M抑制细胞增殖可能与诱导细胞凋亡有关。进一步研究发现,经地钱素M处理后的PC3细胞的形态发生显著变化,Giemsa染色和PI/Hoechest33342双染结果显示,细胞变圆,脱落明显,且细胞形态出现凋亡细胞的特征性改变,DAPI染色观察到细胞核发生明显的凋亡性变化。Westernblotting结果显示,地钱素M处理后,PC3细胞内的抗凋亡蛋白Bcl2的表达下调,而促凋亡蛋白Bax的表达明显增高,其Bax/Bcl2的比率显著增加。由于Caspase在细胞凋亡中发挥重要作用,我们的检测结果表明,地钱素M能够激活PC3细胞中Caspase 3活性,促进多聚ADP-核糖聚合酶(PARP)的剪切,说明地钱素M可能以Caspase依赖的方式诱导PC3细胞凋亡。而经地钱素M处理后,细胞内Caspase 9和Caspase 4的活性增加,但Caspase 8活性并无明显改变,表明线粒体、内质网途径可能参与地钱素M诱导PC3细胞凋亡的作用,但其机制仍需进一步研究。由于地钱素M与茶多酚的结构相似,后者已被证实具有抑制蛋白酶体的作用,因而我们检测地钱素M对蛋白酶体的酶解活性的影响。检测结果显示,地钱素M在体外显著抑制纯化的20S蛋白酶体的糜蛋白酶样活性、多肽／谷氨酰水解酶活性,其抑制作用随浓度的增加而增强。而经地钱素M作用后PC3细胞内糜蛋白酶样活性、多肽／谷氨酰水解酶活性随着地钱素M作用浓度的增加、作用时间的延长而显著降低,表明地钱素M具有明显的抑制蛋白酶体活性的作用。因此,地钱素M可能是通过抑制蛋白酶体活性发挥了诱导PC3细胞凋亡的作用。
总之,本研究结果表明,双联苄类化合物地钱素M具有抑制激素非依赖性前列腺癌PC3细胞增殖、引起细胞周期阻滞、诱导细胞凋亡的作用。其作用机制可能是通过抑制蛋白酶体活性诱导细胞凋亡,线粒体和内质网可能参与其诱导凋亡的过程。本研究首次报道一种新型的蛋白酶体抑制剂,为天然的双联苄化合物的进一步开发利用提供了新的思路。
Prostate cancer (prostate cancer, PCa) is the most common malignant cancer and the second cause of death in men in Europe and America countries. Recently, prostate cancer has become more common in our country.
PCa is an androgen-dependent tumor, and androgen ablation serves as an standard treatment for patients who present advanced, androgen-dependent metastatic PCa for many years. However, the high failure rate for castration causes progresses to hormone refractory prostate cancer (HRPC), that is resistant to radiation, surgery, and chemotherapy. It is the major cause of the mortality associated with HRPC. Despite numerous advances including aberrant regulation of androgen/androgen receptor leading to constitutive active androgen receptor, activation of MAPK, PI3K/Akt, JAK-STAT signaling pathways which contribute cell proliferation in addition to androgen stimuli in PCa, abnormal control of apoptosis cascade such as alterations of the expression of Bcl2, Bax resulting in apoptosis resistance, the high mortality rate occurs because the treatment of this stage of PCa has been largely ineffective, eventually the patients would die of an HRPC.
To date, chemotherapy is still a common clinical strategy for HRPC. For example, the pacitaxel, Present clinical drugs has their own chemical and biological characteristics, yet treatment outcomes are not satisfactory. The mechanism includes two aspects. (1)Patients with advanced prostate cancers undergo distant metastasis, and have many complications. (2) Despite rapid symptoms improved and lifetime prolonged, serious side effects of chemical drugs cause damage of body, repress immune system and result in poor prognosis. For example, Chepatotoxicity though with the advantages of wide spectrum anti-tumor effects and strong effects; Cyclophosphamide exhibits bone marrow depression; docetaxel triggers cancer cells to produce drug resistance. Therefore, it is required urgently to find novel drugs with high effects and low toxicity to treat the androgen-independent PCas.
Ubiquitin-proteasome system (UPS), the major proteolytic enzyme system in mammalian cells, plays critical roles in basic cellular functional process, including proliferation, differentiation and proliferation, and so on. As a proteolytic enzyme complex, UPS is an ATP dependent proteolytic complex, which is comprised of 20 S catalytic particle, 11S regulatory factor and two 19S regulatory particles. Maintaining the activity of UPS is greatly involved in keeping the maintenance of cell basic functions. UPS precisely degrades target proteins in mammalian cells, and then participates in the regulation of gene transcription and cell cycle, as well as in the cellular physical process which includes receptor-mediated endocytosis and antigen processing. Therefore, UPS will be an important target for drug treatment. Recent studies have validated that malignant tumors are more sensitive to UPS inhibitors compared to non-malignant tumors. Our study found that Marchantin M could inhibit the activity of UPS and the proliferation of PC3 cells.
At present, natural compound with anticancer constituents is highly concerned. liverworts contains a number of natural compound, including terpenoid, phenols and bisbibenzyls, among which bisbibenzyls with unique chemical structure and wide biological property receive great attention. We screened many bisbibenzyls candidates and found that Marchantin M exhibited inhibiting effect of proteasome activity and had significantly anticancer activity of PCa PC3 cells. The mechanism by which Marchantin M inhibited proliferation of PC3 cells and activity of proteasome was discussed in our study.
Cell proliferation assay, a method to test antitumor activity of Marchantin M, showed that Marchantin M remarkably repressed proliferation of adenocarcinoma cells including breast cancer MCF-7 cells, prostate cancer LNCaP, DU145 and PC3 cells. This effect was especially evident in androgen-independent PCa PC3 cells. Additionally, Marchantin M was of low toxicity in normal human retinal epithelium cells. Marchantin M significantly depressed PC3 cells in the concentration of 20μmol/L. Yet, in this concentration, it hardly had repression in hTERT-RPE1. Cell morphological alterations were observed by Gimsa staining and double staining (PI and Hoechest33342) methods. The results indicated that cells remarkably dropped off and cell morphology exhibited the characteristic changes of apoptosis. Next, we tested cell cycle by flow cytometry and western blotting, and found that apoptosis rate increased dramatically compared to the control group and cells were apparently arrested in G0/G1 phase. Changes of apoptosis related proteins by western bolting exhibited that anti-apoptosis protein Bcl2 was down-regulated whereas pro-apoptosis was significantly increased in treatment group. In the concentration of 10μmol/L, we found activation of Caspase 3, cleavage of PARP, and activation of Caspase 4 and Caspase 9. However, obvious activation of Caspase 8 was not observed. These data indicated that Marchantin M inhibited proliferation of PC3 cells by inducing apoptosis of them. Further study was made on the mechanism of apoptosis in PC3 cells by Marchantin M. Structure of Marchantin M has been validated to be similar to a proteasome inhibitor (tea polyphenol) in recent studies. So we supposed that Marchantin M might induce apoptosis of PC3 cells by inhibiting activity of proteasome. we detected activity of proteasome and found Marchantin M could inhibit enzymolysis activity of proteasome in vitro and also repress the enzymolysis activity of proteasome for total proteins in PC3 cells. This was consistent with our hypothesis.
In summary, our study showed that Marchantin M could significantly inhibit proliferation of androgen-independent PCa PC3 cells and increase apoptosis of PC3 cells. The potential mechanism is that repression of UPS lead to cell apoptosis. In our study, the novel proteasome inhibitor to cope with PCa will give new hope for treatment of human PCa.
引文
1.甘琳,李鸣.雄激素非依赖性前列腺癌发病机制的研究进展.中华外科杂志.2007,45(8)：562-564.
2.陈永胜,李翼飞.雄激素非依赖性前列腺癌生物学机制的研究进展.哈尔滨医科大学学报.2004,38(4)：403-404.
3. Roberts PJ, Der CJ. Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene.2007,26:3291-3310.
4. Kinkade CW, Castillo-Martin M, Puzio-Kuter A, et al. Targeting AKT/mTOR and ERK/MAPK signaling inhibits hormone-refractory prostate cancer in a preclinical mouse model. J Clin Invest.2008,118:3051-3064.
5. Yamanaka K, Rocchi P, Miyake H, et al. Induction of apoptosis and enhancement of chemosensitivity in human prostate cancer LNCaP cells using bispecific antisense oligonucleotide targeting Bcl-2 and Bcl-xL genes. BJU International. 2006,97:1300-1308.
6. Montagut C, Rovira A, Albanell J. The proteasome: a novel target for anticancer therapy. Clin Transl Oncol.2006,8(5):313-317.
7. Zavrski I, Kleeberg L, Kaiser M, et al. Proteasome as an emerging therapeutic target in cancer. Curr Pharm Des.2007,13:471-485.
8. Bailly C. Ready for a comeback of natural products in oncology. Biochem Pharmacol.2008,77(9):1447-1457.
9. Asakawa Y, Toyota M, Taira Z, et al. Riccardin A and Riccardin B, Two Novel Cyclic Bibenzyls Possessing Cytotoxicity from the Liverwort Riccardia multifida (L.) S. Gray. J. Org. Chem.1983,48:2164-2167.
10. Scher JM, Burgess EJ, Lorimer SD, et al. A cytotoxic sesquiterpene and unprecedented sesquiterpene bisbibenzyl compounds from the liverwort Schistochila glaucescens. Tetrahedron.2002,58:7875-7882.
11. Nagashima F, Momosaki S, Watanabe Y, et al. Terpenoids and aromatic compounds from six liverworts. Phytochemistry.1996,41:207-211.
12. Qu JB, Xie CF, Lou HX, et al. Antifungal dibenzofuran bis(bibenzyl)s from the liverwort Asterella angusta. Phytochemistry.2007,68:1767-1774.
13. Xie CF, Qu JB, Wu XZ, et al. Antifungal macrocyclic bis(bibenzyls) from the Chinese liverwort Ptagiochasm intermedlum L. Nat Prod Res.2010,24(6): 515-520.
14. Niu C, Qu JB, Lou HX. Antifungal bis[bibenzyls] from the Chinese liverwort Marchantia polymorpha L. Chem Biodivers.2006,3:34-40.
15. Wang FQ, Lou HX. Chemical studies on the constituents of Plagiochasm intermedium L. Acta Pharmacol Sin.2000,35:587-591.
16. Landis SH, Murray T, Bolden S, et al. Cancer statistics. Cancer J Urol.1994, 152:1677-1678.
17. Debes JD, Tindall DJ. Mechanisms of androgen-refractory prostate cancer. N J Med.2004,351(15):1488-1490.
18. GrossmannⅧ, Huang H, Tindall DJ. Androgen receptor signaling in androgen-refractory prostate cancer. J Natl Cancer Inst.2001,93(22):1687-1 697.
19. Wilson JD, Griffin JE, George FW, et al. The role of gonadal steroids in sexual differentiation. Recent Prog Horm Res.1981,37:1-9.
20. Chen CD, Welsbie DS, Tran C, et al. Molecular determinants of resistance to anti-androgen therapy. Nat Med.2004,10:33-39.
21. Tomlins SA, Mehra R, Rhodes DR, et al. Integrative molecular concept modeling of prostate cancer progression. Nat Genet.2007,39:41-51.
22. Kousteni S, Bellido T, Plotkin LI, et al. Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors:dissociation from transcriptional activity. Cell.2001,104:719-730.
23. Hudes G, Ross E, Dugan, et al. Improved survival for patients with hormone-refractory prostate cancer receiving estramustine-based antimicrotubule
therapy:final report of a hoosier oncology group and fox chase network phase III trial comparing vinbalastine and vinblastine plus oral estramustine phosphate. Proc Am Soc Clin Oncol.2002,21(7):177-182.
24. pienta KJ, Fisher EL, Eisenberger MA. A phase II trial of estramustine and etoposide in hormone refractory prostate cancer: A southwest oncology Group trial (SWOG 9407). Prostate.2001,46:257-261.
25. Yagoda A, Petrylak D. Cytotoxic chemotherapy for advanced hormone-resistant prostate cancer. Cancer.1993,71:1098-1109.
26. Tannock IF, Osoba D, Stockler MR, et al. Chemotherapy with mitoxantrone plus prednisone or prednisone alone for symptomatic hormone-resistant prostate cancer:a Canadian randomized trial with palliative end points. J Clin Oncol.1996, 14:1756-1764.
27.秦自科,杨光伟,周芳坚等.晚期激素非依赖性前列腺癌临床化疗效果分析.中华泌尿外科杂志.2007,28：120-123.
28. Kolvenbag GTCM, Iversen D, Newling DWW. Antiandrogen monotherapy. A new form of treatment for patients with prostate cancer. Urology.2001,58(2): 16-22.
29. Oh WK, Kantoff PW. Management of hormone refractory prostate cancer: current standards and future prospect. J Urol.1998,160(4):1220-1229.
30. Rosenberg JE, Galsky MD, Rohs NC, et al. A retrospective evaluation of second line chemotherapy response in hormone refractory prostate carcinoma:second line taxane based therapy after first-line epothilone B analog ixabepilone (BM S247550) therapy. Cancer.2006,106(7):58-62.
31. Strous GJ, Govers R. The ubiquitin-proteasome system and endocytosis. J Cell Sci.1999,112:1417-1423
32. Wu HJ, Zhang ZG. Ubiquitin-Proteasome pathway and its significance. J Int Patho Clinic Med.2006,26(1):7-10.
33. Strick land E, Hakala K, Thomas PJ, et al. Recognition of misfolding proteins by PA700, the regulatory subcomplex of the 26 S proteasome. J Biol Chem.2000, 275 (8):5565-5572.
34. Sakata N, Dixon JL. Ubiquitin-proteasome-dependent degradation of apolipoprotein B100 in vitro. Biochim Biophys Acta.1999,1437 (1):71-79.
35. Murray RZ, Norbury C. Proteasome inhibitors as anti-cancer agents. Anti-cancer Drugs.2000,11(6):407-441.
36. Kerr JF, Wyllie AH, Currie AR. Apoptosis:a basic biological phenomenon with wild-ranging implications in tissue kinetics. Br J Cancer.1972,26:239-257.
37. Nakamura K, Bossy-Wetzel E, Burns K,et al. Changes in endoplasmic reticulum luminal environment affect cell sensitivity to apoptosis. J Cell Biol.2000,150(4): 731-740.
38. Rao RV, Hermel E, Castrol-Obregon S, et al. Coupling endoplasmic reticulum stress to the cell death program. Mechanism of Caspase activation. J Cell Biol. 2001,276(36):33869-33874.
39. Yamagishi S, Bando Y, Imaizumi K, et al. Involvement of Caspase-4 in endoplasmic reticulum stress-induced apoptosis and Abeta-induced cell death. J. Cell Biol.2004,165:347-356.
40. Chien MM, Zahradka KE, Newell M, el al. Fas-induced B cell apoptosis requires an increase in flee cytosolic magnesium as an early event. J Biol. Chem. 1999,274(11):7059-7066.
41. Patterson SD, Spahr CS, Daugas E. Mass spectrometric identification of proteins released from mitochondria undergoing permeability transition. Cell Death Differ. 2000,7(2):137-144.
42. Singh RP, Dhanalakshmi S, Agarwal R. Phytochemicals as cell cycle modulators: a less toxic approach in halting human cancers. Cell Cycle.2002,1:156-161.
43. Lee KH. Anticancer drug design based on plant-derived natural products. J Biomed Sci.1999,6:236-250.
44. Baek SH, Phipps RK, Perry NB. Antimicrobial chlorinated bibenzyls from the liverwort Riccardia marginata. J Nat Prod.2004,67:718-720.
45. Scher JM, Speakman JB, Zapp J, et al. Bioactivity guided isolation of antifungal compounds from the liverwort Bazzania trilobata (L.) S.F. Gray. Phytochemistry. 2004,65:2583-2588.
46. Kodama M, Shiobara Y, Sumitomo H, et al. Total Syntheses of marchantin A and riccardin B, Cytotoxic Bis(bibenzyls) from Liverworts. J Org Chem.1988,53: 72-77.
47. Shi YQ, Zhu CJ, Yuan HQ, et al. Marchantin C, a novel microtubule inhibitor from liverwort with anti-tumor activity both in vivo and in vitro. Cancer Lett. 2008,276:160-170.
48. Shi YQ, Liao YX, Qu XJ, et al. Marchantin C, a macrocyclic bisbibenzyl, induces apoptosis of human glioma A172 cells. Cancer Lett.2008,262:173-182.
49. Shi YQ, Qu XJ, Liao YX, et al. Reversal effect of a macrocyclic bisbibenzyl plagiochin E on multidrug resistance in adriamycin-resistant K562/A02 cells. Eur J Pharm.2008,584:66-71.
50. Asakawa Y, Bors W, Franck U, et al. Effect of marchantins and related compounds on 5-lipoxygenase and cyclooxygenase and their antioxidant properties: a structure-activity relationship study. Phytomedicine.1995,2: 113-117.
51. Harada K, Supriatno, Yamamoto S, et al. Cepharanthine exerts antitumor activity on oral squamous cell carcinoma cell lines by induction of p27~(Kip1). Anticancer Res.2003,23(2B):1441-1448.
52. Yu ZY, Qin XL, Gu YY, et al. Prodrugs of Fluoro-Substituted Benzoates of EGC as Tumor Cellular Proteasome Inhibitors and Apoptosis Inducers. Int J Mol Sci. 2008,9(6):951-961.
53. McConkey DJ, Greene G, Pettaway CA. Apoptosis resistance increases with metastatic potential in cells of the human LNCaP prostate carcinoma line. Cancer Res.1996,56:5594-5599.
54. Lebedeva I, Rando R, Ojwang J, et al. Bcl-xL in prostate cancer cells:effect of overexpression and down-regulation on chemotherapy. Cancer Res.2000,60: 6052-6060.
55. Fizazi K, Martinez LA, Sikes CR, et al.The association of p21~((WAF-1/CIP1))with progression to androgen-independent prostate cancer. Clin Cancer Res.2002,8(3): 775-781.
56. Don MJ, Chang YH, Chen KK, et al. Induction of CDK inhibitors (p21~((WAF-1))and p27~((Kip1))and Bak in the beta-lapachone-induced apoptosis of human prostate
cancer cells. Mol Pharmacol.2001,59(4):784-794.
57. Gotoh A, Shirakawa T, Wada Y, et al. The growth inhibitory effect of p21 adenovirus on androgen-dependent and independent human prostate cancer cells. BJU Int.2003,92(3):314-318.
58. Spano JP, Bay JO, Blay JY,et al. Proteasome inhibition: a new approach for the treatment of malignancies. Bull Cancer.2005,92(11):E61-E66,945-952.
59. Burger AM, Seth AK. The ubiquitin-mediated protein degradation pathway in cancer:therapeutic implications. Eur J Cancer.2004,15(40):2217-2229.
60. Pickart CM, Eddins MJ. Ubiquitin: structure, functions mechanisms. Biochim Biophys Acta.2004,1695(1-3):55-72.
61. Hideshima T, Richardson P, Chauhan D, et al. The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells. Cancer Res.2001,61:3071-3076.
62. Jackson G, Einsele H, Moreau P, et al. Bortezomib, a novel proteasome inhibitor, in the treatment of hematologic malignancies. Cancer Treat Rev.2005,8(31): 591-602.
63. Kenneth C, Anderson. The clinical potential of proteasome inhibition. Eur J Can Sup.2004,6(2):3-6.
64. Yang H, Dou QP. Celestrol, a trierpene extracted from the Chinese "Thunder of God Vine" is a potent proteasome inhibitor and suppresses human prostate cancer growth in nude mice. Cancer Res.2006,66(9):4758-4765.
65. Drexler HC. Activation of the cell death program by inhibition of proteasome function. Proc.Natl Acad.Sci. USA.1997,94:855-860.
66. Drexler HC, Risav W, Konerding MA. Inhibition of proteasome function induces programmed cell death in proliferating endothelial cells. FASEB J.2000,14: 65-77.
67. Kudo Y, Takata T, Ogawa I, et al. p27Kipl accumulation by inhibition of proteasome function induces apoptosis in oral squamous cell carcinoma cells. Clin Cancer Res.2002,6:916-923.
68. Bogner C, Schneller F, Hipp S. Cycling B-CLL cells are highly susceptible to inhibition of the proteasome:involvement of p27, early D-type cyclins, Bax, and Caspase dependent and independent pathways. Exp Hematol.2003,31: 218-225.
2.陈永胜,李翼飞.雄激素非依赖性前列腺癌生物学机制的研究进展.哈尔滨医科大学学报.2004,38(4)：403-404.
3. Roberts PJ, Der CJ. Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene.2007,26:3291-3310.
4. Kinkade CW, Castillo-Martin M, Puzio-Kuter A, et al. Targeting AKT/mTOR and ERK/MAPK signaling inhibits hormone-refractory prostate cancer in a preclinical mouse model. J Clin Invest.2008,118:3051-3064.
5. Yamanaka K, Rocchi P, Miyake H, et al. Induction of apoptosis and enhancement of chemosensitivity in human prostate cancer LNCaP cells using bispecific antisense oligonucleotide targeting Bcl-2 and Bcl-xL genes. BJU International. 2006,97:1300-1308.
6. Montagut C, Rovira A, Albanell J. The proteasome: a novel target for anticancer therapy. Clin Transl Oncol.2006,8(5):313-317.
7. Zavrski I, Kleeberg L, Kaiser M, et al. Proteasome as an emerging therapeutic target in cancer. Curr Pharm Des.2007,13:471-485.
8. Bailly C. Ready for a comeback of natural products in oncology. Biochem Pharmacol.2008,77(9):1447-1457.
9. Asakawa Y, Toyota M, Taira Z, et al. Riccardin A and Riccardin B, Two Novel Cyclic Bibenzyls Possessing Cytotoxicity from the Liverwort Riccardia multifida (L.) S. Gray. J. Org. Chem.1983,48:2164-2167.
10. Scher JM, Burgess EJ, Lorimer SD, et al. A cytotoxic sesquiterpene and unprecedented sesquiterpene bisbibenzyl compounds from the liverwort Schistochila glaucescens. Tetrahedron.2002,58:7875-7882.
11. Nagashima F, Momosaki S, Watanabe Y, et al. Terpenoids and aromatic compounds from six liverworts. Phytochemistry.1996,41:207-211.
12. Qu JB, Xie CF, Lou HX, et al. Antifungal dibenzofuran bis(bibenzyl)s from the liverwort Asterella angusta. Phytochemistry.2007,68:1767-1774.
13. Xie CF, Qu JB, Wu XZ, et al. Antifungal macrocyclic bis(bibenzyls) from the Chinese liverwort Ptagiochasm intermedlum L. Nat Prod Res.2010,24(6): 515-520.
14. Niu C, Qu JB, Lou HX. Antifungal bis[bibenzyls] from the Chinese liverwort Marchantia polymorpha L. Chem Biodivers.2006,3:34-40.
15. Wang FQ, Lou HX. Chemical studies on the constituents of Plagiochasm intermedium L. Acta Pharmacol Sin.2000,35:587-591.
16. Landis SH, Murray T, Bolden S, et al. Cancer statistics. Cancer J Urol.1994, 152:1677-1678.
17. Debes JD, Tindall DJ. Mechanisms of androgen-refractory prostate cancer. N J Med.2004,351(15):1488-1490.
18. GrossmannⅧ, Huang H, Tindall DJ. Androgen receptor signaling in androgen-refractory prostate cancer. J Natl Cancer Inst.2001,93(22):1687-1 697.
19. Wilson JD, Griffin JE, George FW, et al. The role of gonadal steroids in sexual differentiation. Recent Prog Horm Res.1981,37:1-9.
20. Chen CD, Welsbie DS, Tran C, et al. Molecular determinants of resistance to anti-androgen therapy. Nat Med.2004,10:33-39.
21. Tomlins SA, Mehra R, Rhodes DR, et al. Integrative molecular concept modeling of prostate cancer progression. Nat Genet.2007,39:41-51.
22. Kousteni S, Bellido T, Plotkin LI, et al. Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors:dissociation from transcriptional activity. Cell.2001,104:719-730.
23. Hudes G, Ross E, Dugan, et al. Improved survival for patients with hormone-refractory prostate cancer receiving estramustine-based antimicrotubule
therapy:final report of a hoosier oncology group and fox chase network phase III trial comparing vinbalastine and vinblastine plus oral estramustine phosphate. Proc Am Soc Clin Oncol.2002,21(7):177-182.
24. pienta KJ, Fisher EL, Eisenberger MA. A phase II trial of estramustine and etoposide in hormone refractory prostate cancer: A southwest oncology Group trial (SWOG 9407). Prostate.2001,46:257-261.
25. Yagoda A, Petrylak D. Cytotoxic chemotherapy for advanced hormone-resistant prostate cancer. Cancer.1993,71:1098-1109.
26. Tannock IF, Osoba D, Stockler MR, et al. Chemotherapy with mitoxantrone plus prednisone or prednisone alone for symptomatic hormone-resistant prostate cancer:a Canadian randomized trial with palliative end points. J Clin Oncol.1996, 14:1756-1764.
27.秦自科,杨光伟,周芳坚等.晚期激素非依赖性前列腺癌临床化疗效果分析.中华泌尿外科杂志.2007,28：120-123.
28. Kolvenbag GTCM, Iversen D, Newling DWW. Antiandrogen monotherapy. A new form of treatment for patients with prostate cancer. Urology.2001,58(2): 16-22.
29. Oh WK, Kantoff PW. Management of hormone refractory prostate cancer: current standards and future prospect. J Urol.1998,160(4):1220-1229.
30. Rosenberg JE, Galsky MD, Rohs NC, et al. A retrospective evaluation of second line chemotherapy response in hormone refractory prostate carcinoma:second line taxane based therapy after first-line epothilone B analog ixabepilone (BM S247550) therapy. Cancer.2006,106(7):58-62.
31. Strous GJ, Govers R. The ubiquitin-proteasome system and endocytosis. J Cell Sci.1999,112:1417-1423
32. Wu HJ, Zhang ZG. Ubiquitin-Proteasome pathway and its significance. J Int Patho Clinic Med.2006,26(1):7-10.
33. Strick land E, Hakala K, Thomas PJ, et al. Recognition of misfolding proteins by PA700, the regulatory subcomplex of the 26 S proteasome. J Biol Chem.2000, 275 (8):5565-5572.
34. Sakata N, Dixon JL. Ubiquitin-proteasome-dependent degradation of apolipoprotein B100 in vitro. Biochim Biophys Acta.1999,1437 (1):71-79.
35. Murray RZ, Norbury C. Proteasome inhibitors as anti-cancer agents. Anti-cancer Drugs.2000,11(6):407-441.
36. Kerr JF, Wyllie AH, Currie AR. Apoptosis:a basic biological phenomenon with wild-ranging implications in tissue kinetics. Br J Cancer.1972,26:239-257.
37. Nakamura K, Bossy-Wetzel E, Burns K,et al. Changes in endoplasmic reticulum luminal environment affect cell sensitivity to apoptosis. J Cell Biol.2000,150(4): 731-740.
38. Rao RV, Hermel E, Castrol-Obregon S, et al. Coupling endoplasmic reticulum stress to the cell death program. Mechanism of Caspase activation. J Cell Biol. 2001,276(36):33869-33874.
39. Yamagishi S, Bando Y, Imaizumi K, et al. Involvement of Caspase-4 in endoplasmic reticulum stress-induced apoptosis and Abeta-induced cell death. J. Cell Biol.2004,165:347-356.
40. Chien MM, Zahradka KE, Newell M, el al. Fas-induced B cell apoptosis requires an increase in flee cytosolic magnesium as an early event. J Biol. Chem. 1999,274(11):7059-7066.
41. Patterson SD, Spahr CS, Daugas E. Mass spectrometric identification of proteins released from mitochondria undergoing permeability transition. Cell Death Differ. 2000,7(2):137-144.
42. Singh RP, Dhanalakshmi S, Agarwal R. Phytochemicals as cell cycle modulators: a less toxic approach in halting human cancers. Cell Cycle.2002,1:156-161.
43. Lee KH. Anticancer drug design based on plant-derived natural products. J Biomed Sci.1999,6:236-250.
44. Baek SH, Phipps RK, Perry NB. Antimicrobial chlorinated bibenzyls from the liverwort Riccardia marginata. J Nat Prod.2004,67:718-720.
45. Scher JM, Speakman JB, Zapp J, et al. Bioactivity guided isolation of antifungal compounds from the liverwort Bazzania trilobata (L.) S.F. Gray. Phytochemistry. 2004,65:2583-2588.
46. Kodama M, Shiobara Y, Sumitomo H, et al. Total Syntheses of marchantin A and riccardin B, Cytotoxic Bis(bibenzyls) from Liverworts. J Org Chem.1988,53: 72-77.
47. Shi YQ, Zhu CJ, Yuan HQ, et al. Marchantin C, a novel microtubule inhibitor from liverwort with anti-tumor activity both in vivo and in vitro. Cancer Lett. 2008,276:160-170.
48. Shi YQ, Liao YX, Qu XJ, et al. Marchantin C, a macrocyclic bisbibenzyl, induces apoptosis of human glioma A172 cells. Cancer Lett.2008,262:173-182.
49. Shi YQ, Qu XJ, Liao YX, et al. Reversal effect of a macrocyclic bisbibenzyl plagiochin E on multidrug resistance in adriamycin-resistant K562/A02 cells. Eur J Pharm.2008,584:66-71.
50. Asakawa Y, Bors W, Franck U, et al. Effect of marchantins and related compounds on 5-lipoxygenase and cyclooxygenase and their antioxidant properties: a structure-activity relationship study. Phytomedicine.1995,2: 113-117.
51. Harada K, Supriatno, Yamamoto S, et al. Cepharanthine exerts antitumor activity on oral squamous cell carcinoma cell lines by induction of p27~(Kip1). Anticancer Res.2003,23(2B):1441-1448.
52. Yu ZY, Qin XL, Gu YY, et al. Prodrugs of Fluoro-Substituted Benzoates of EGC as Tumor Cellular Proteasome Inhibitors and Apoptosis Inducers. Int J Mol Sci. 2008,9(6):951-961.
53. McConkey DJ, Greene G, Pettaway CA. Apoptosis resistance increases with metastatic potential in cells of the human LNCaP prostate carcinoma line. Cancer Res.1996,56:5594-5599.
54. Lebedeva I, Rando R, Ojwang J, et al. Bcl-xL in prostate cancer cells:effect of overexpression and down-regulation on chemotherapy. Cancer Res.2000,60: 6052-6060.
55. Fizazi K, Martinez LA, Sikes CR, et al.The association of p21~((WAF-1/CIP1))with progression to androgen-independent prostate cancer. Clin Cancer Res.2002,8(3): 775-781.
56. Don MJ, Chang YH, Chen KK, et al. Induction of CDK inhibitors (p21~((WAF-1))and p27~((Kip1))and Bak in the beta-lapachone-induced apoptosis of human prostate
cancer cells. Mol Pharmacol.2001,59(4):784-794.
57. Gotoh A, Shirakawa T, Wada Y, et al. The growth inhibitory effect of p21 adenovirus on androgen-dependent and independent human prostate cancer cells. BJU Int.2003,92(3):314-318.
58. Spano JP, Bay JO, Blay JY,et al. Proteasome inhibition: a new approach for the treatment of malignancies. Bull Cancer.2005,92(11):E61-E66,945-952.
59. Burger AM, Seth AK. The ubiquitin-mediated protein degradation pathway in cancer:therapeutic implications. Eur J Cancer.2004,15(40):2217-2229.
60. Pickart CM, Eddins MJ. Ubiquitin: structure, functions mechanisms. Biochim Biophys Acta.2004,1695(1-3):55-72.
61. Hideshima T, Richardson P, Chauhan D, et al. The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells. Cancer Res.2001,61:3071-3076.
62. Jackson G, Einsele H, Moreau P, et al. Bortezomib, a novel proteasome inhibitor, in the treatment of hematologic malignancies. Cancer Treat Rev.2005,8(31): 591-602.
63. Kenneth C, Anderson. The clinical potential of proteasome inhibition. Eur J Can Sup.2004,6(2):3-6.
64. Yang H, Dou QP. Celestrol, a trierpene extracted from the Chinese "Thunder of God Vine" is a potent proteasome inhibitor and suppresses human prostate cancer growth in nude mice. Cancer Res.2006,66(9):4758-4765.
65. Drexler HC. Activation of the cell death program by inhibition of proteasome function. Proc.Natl Acad.Sci. USA.1997,94:855-860.
66. Drexler HC, Risav W, Konerding MA. Inhibition of proteasome function induces programmed cell death in proliferating endothelial cells. FASEB J.2000,14: 65-77.
67. Kudo Y, Takata T, Ogawa I, et al. p27Kipl accumulation by inhibition of proteasome function induces apoptosis in oral squamous cell carcinoma cells. Clin Cancer Res.2002,6:916-923.
68. Bogner C, Schneller F, Hipp S. Cycling B-CLL cells are highly susceptible to inhibition of the proteasome:involvement of p27, early D-type cyclins, Bax, and Caspase dependent and independent pathways. Exp Hematol.2003,31: 218-225.