甲异靛抗慢性髓性白血病和结肠癌HT-29的活性及作用机制研究
摘要
甲异靛(meisoindigo)是靛玉红类双吲哚化合物,是目前临床治疗慢性髓性白血病(chronic myelogenous leukemia,CML)的重要化疗药物。靛玉红是从中药复方当归龙荟丸的青黛中分离提取出的有效成分,但是靛玉红溶解度差,胃肠道不良反应比较严重。中国医学科学院中国协和医科大学药物所在靛玉红的结构基础上合成了靛玉红的衍生物甲异靛。甲异靛溶解度和临床疗效均比靛玉红好,而且甲异靛的不良反应也相对较轻。在临床上甲异靛已经取代了靛玉红,成为治疗慢性髓性白血病重要的化疗药物。以前的文献报道甲异靛在体外能强烈抑制W256细胞DNA和RNA的生物合成、抑制细胞内微管的聚合、抗血管新生作用和诱导细胞凋亡等,吴英理等曾报道甲异靛对慢性髓性白血病特征性的BCR/ABL蛋白的表达和活性没有影响,提示甲异靛治疗慢性髓性白血病的作用机制可能不是通过与ATP竞争性地抑制TPK活性中心而抑制BCR/ABL的酪氨酸激酶活性。因此,需要进一步研究甲异靛治疗慢性髓性白血病的作用机制。
目前,大量研究结果表明,靛玉红类化合物对糖原合成酶激酶GSK-3β(glycogen-synthase kinase,GSK)和细胞周期素依赖激酶CDK(cyclin-dependentkinase,CDK)有抑制作用,GSK-3β和CDKs都是丝/苏氨酸蛋白激酶,二者与丝裂原激活激酶(mitogen-activated kinase)及CDK样激酶(CDK-like kinase)同属于CMGC(Cyclin-dependent kinase,Map kinase,Glycogen synthase kinase and Cdk likekinases,CMGC)蛋白家族。GSK-3β是细胞内多种重要信号通路中重要的调控蛋白。GSK-3β是一种多功能的蛋白激酶,不仅参与细胞内的糖代谢,而且与细胞的分化、增殖和凋亡有密切关系,参与细胞内胰岛素信号通路、Wnt信号通路和NF-κB信号通路等。GSK-3β与人类糖尿病、神经退行性疾病、双向情感障碍和肿瘤等疾病的发生有密切的关系。肿瘤细胞一个重要特征是细胞周期调控机制的破坏导致细胞无限制地增殖。CDK和细胞周期素(cyclin)是细胞周期重要的调控蛋白,对细胞周期的准确调控起着重要作用,细胞周期的准确调控对细胞的增殖、分化和凋亡十分重要。当细胞周期调节出现异常时,在人类会导致各种疾病的发生如肿瘤、神经退行性疾病、心血管疾病和病毒感染等。因此,GSK-3β和CDK已经成为目前抗肿瘤药物研究的重要靶点。
GSK-3β是Wnt信号通路中重要的调控蛋白,Wnt信号通路与肿瘤的发生有密切关系,特别是在结肠癌中发现Wnt信号通路中APC(adenomatous polyposis coli,APC)、β-粘连蛋白(β-catenin)等多种蛋白异常,目前认为Wnt信号通路调节异常是结肠癌的重要发病机制之一。因此,GSK-3β抑制剂可能是治疗结肠癌的重要潜在药物之一。
基于以前对甲异靛的作用研究和目前分子生物学对靛玉红类化合物的研究进展,我们对甲异靛治疗慢性髓性白血病的作用机制和对实体瘤的抗瘤活性及作用机制进行了研究。
甲异靛治疗慢性髓性白血病机制研究
甲异靛在体外对K562细胞和HL60细胞增殖有明显的抑制作用,其IC_(50)分别为1.43μmol/L和5.60μmol/L,甲异靛能使K562细胞和HL60细胞阻滞在S期,诱导细胞凋亡,核小体DNA断裂,出现典型的DNA ladder条带。甲异靛能使HL60细胞Wnt信号通路中β-catenin和c-myc基因表达下降,而对K562细胞中β-catenin和c-myc基因表达没有明显影响,甲异靛使Writ信号通路中p-GSK-3β(Ser9)、β-catenin和c-myc蛋白表达下降,GSK-3β(Ser9)蛋白表达增加。甲异靛能诱导K562细胞和HL60细胞凋亡相关蛋白p53和Bax蛋白表达增加,bcl-2蛋白表达下降。K562细胞和HL60细胞中CDK2、cyclinA、cyclinB蛋白表达减少。这些结果提示甲异靛可能是通过竞争性抑制GSK-3β(Ser 9)磷酸化,使细胞内GSK-3β(Ser9)和GSK-3βTyr216磷酸化的平衡破坏,GSK-3β活性相对增强。一方面GSK-3β活性相对增强后胞浆内的β-catenin磷酸化增加,胞浆内磷酸化β-catenin泛素化后被蛋白酶体降解,胞浆内磷酸化β-catenin含量下降,进入细胞核内β-catenin相对减少,因此,与细胞分化和增殖相关的基因和蛋白如c-myc和cyclinD1等的表达相应减少。另一方面GSK-3β活性增强后,GSK-3β能短暂地与p53结合并激活p53,通过p53线粒体凋亡途径,即p53-Bax线粒体凋亡途径。p53发生移位使线粒体内抗凋亡蛋白bcl-2和促凋亡蛋白Bax平衡破坏,导致细胞色素c释放,诱发细胞内促凋亡因子不可逆地释放而激发细胞凋亡。细胞周期调控的主要蛋白是CDKs和cyclins,CDK是细胞周期调节和RNA转录重要调控蛋白。甲异靛使K562细胞和HL60细胞CDK2蛋白和cyclinA蛋白表达减少,而p53蛋白和p21蛋白表达增加。这可能是甲异靛通过抑制CDK2蛋白活性,从而使K562细胞和HL60细胞在S期受到阻滞。
甲异靛对结肠癌HT-29抗肿瘤活性及机制研究
体外筛选结果表明,甲异靛在体外对乳腺癌细胞MCF-7、非小细胞肺癌A549和HT-29等多种实体瘤细胞增殖都有明显的抑制作用。在实体瘤中对HT-29细胞的抑制活性最强。甲异靛能抑制HT-29细胞生长和集落形成。使HT-29细胞阻滞在G2/M期,诱导细胞凋亡,细胞内核小体DNA断裂出现典型的梯形条带。在基因水平甲异靛能使HT29细胞内β-catenin、c-Myc和bcl-2基因表达降低,Bax基因表达增加。在蛋白水平甲异靛能抑制GSK-3β磷酸化,使Wnt信号通路中pGSK-3β(Ser9)、β-catenin和c-Myc蛋白表达下降,GSK-3β(Ser9)蛋白表达增加。甲异靛抑制周期素依赖激酶(CDK),使cyclinA、cyclinB、p53和p21蛋白表达增加,CDK2蛋白表达减少。甲异靛能使细胞内抗凋亡蛋白bcl-2表达减少,使促凋亡蛋白Bax表达增加。
在人HT-29细胞裸鼠异体移植瘤模型中,甲异靛对HT-29细胞裸鼠异体移植瘤生长有明显抑制作用,与空白对照组比较,甲异靛能较明显抑制裸鼠体内肿瘤的生长,肿瘤抑制率可达到47.60%。瘤组织病理学结果提示,甲异靛对人结肠癌HT-29裸鼠异体移植瘤的生长有一定的抑制作用,随着给药剂量的增加,癌细胞分裂相数有所减少,尤其是大剂量组更为明显。人HT-29细胞裸鼠异体移植瘤组织免疫组化结果表明,甲异靛能抑制裸鼠异体移植瘤组织内GSK-3β(ser9)磷酸化,GSK-3β的活性量增加,而p-GSK-3β蛋白、β-catenin蛋白和c-myc蛋白表达相应减少。
甲异靛对人结肠癌HT-29的体内和体外结果提示,对人结直肠癌HT-29抑制作用可能是通过抑制GSK-3β(Ser9)磷酸化和CDK2活性实现的。甲异靛抑制GSK-3β(Ser9)磷酸化后活性相对增强,胞浆中β-catenin磷酸化增加,胞浆中β-catenin磷酸化后被降解,因此,进入细胞核的β-catenin减少,抑制了细胞核内的c-myc等癌基因的转录和表达。GSK-3β活性相对增加还可引起p53线粒体凋亡途径激活,即GSK-3β-p53-bax途径诱导细胞凋亡。HT-29细胞阻滞在G2/M期,这提示甲异靛可能是通过抑制CDK2活性,CDK2活性降低可直接导致细胞阻滞在G2/M期。另一方面,甲异靛通过抑制GSK-3β使p53蛋白和p21蛋白活性增加,通过p52-p21-CDK2途径,进一步使HT-29细胞阻滞在G2/M期。
总之,甲异靛治疗慢性髓性白血病的机制可能是通过抑制GSK-3β(Ser 9)磷酸化,GSK-3β活性相对增强,一方面使Wnt信号通路中GSK-3β下游β-catenin被磷酸化后降解增加,进入细胞核内β-catenin减少,细胞核内与分化和增殖相关的基因和蛋白如c-myc和cyclinD1等的表达下降。另一方面GSK-3β-p53途径诱导细胞凋亡。甲异靛对实体瘤人结肠癌HT-29的抑制作用也可能是通过抑制GSK-3β(Ser9)磷酸化和CDK2活性实现的。甲异靛在体内和体外对HT-29细胞的抑制作用提示甲异靛在临床上对结肠癌可能有一定的治疗作用。
Meisoindigo,an indirubin derivative,plays an important role in the treatment of chronic myelogenous leukemia(CML).Indirubin is the active indigrent of Danggui Longhui Wan,a mixture of plants that is used in Traditional Chinese Medicine to treatment of CML,but indirubin shows poor solubility and absorption,and exhibites serious side action in gastricintestinal tract.In order to improve indirubin these characteristics,several analogues of indirubin were synthesized by the Institute Materia and Medica,Chinese Academy of Medical Sciences & Peking Union Medical College. One of these analogues,Meisoindigo,shows stronger antitumor activity and only minor toxicity.Meisoindigo was approved for treatment of CML by State Foods and Drugs Administration(SFDA),and Meisoindigo had taken place of indirubin in the treatment of CML in China.Previous studies showed that meisoindigo inhibited DNA and RNA synthesis in W256 cells,prevented microtubule aggregation,inhibited new vascular formation and induced cell apoptosis.Wu Yingli reported that meisoindigo had no effect on BCR/ABL activity and expression,which was the characteristic protein of CML.So it is valuable to study the mechanism of meisoindigo against CML further.
Recently,indirubin and its derivatives were reported to inhibit glycogen-synthase kinase-3(GSK-3) and cyclin-dependent kinase(CDKs) activitives.GSK-3 and CDKs are serine/threonine kinase.Both of them with mitogen-activated kinase and CDK-like kinase belong to CMGC superfamily.GSK-3 is an important regulatory protein in many signal pathways including insulin signal patyway,Wnt signal pathway and NF-κB signal pathway.Not only dose it take part in glycogen synthesis and metabolism,but also participates in cell differentiation,proliferation and apoptosis.The dysregulation of GSK-3βabnormalities associates with diabetes mellitus,Alzheimer's disease,bipolar mood disorder and cancer.CDKs and cyclins are important regulator proteins in cell cycle.The normal cell cycle regulation is a necessary for cell cycle from one phase to the next phase.Cell cycle is relative to cell proliferation and apoptosis.A characteristic of cancer cells is its cell cycle dysregulation,which results in cancer cell permanent proliferation.When cell cycle regulation becomes abnormalities,it will result in cancer, neurodegenerative disease and cardiovascular disease.GSK-3βand CDKs have become the targets of cancer therapy.
GSK-3βplays a central role in Wnt signal pathway which implicats in cell survive and diverse cancers.GSK-3βprotein overexpression has been found in human ovarian, colon and pancreatic carcinoma.In colorectal cancer,many protein mutations including APC,β-catenin were founded.The dysregulation of Wnt signal pathway is one of colon cancer etiopathogenisis.The GSK-3βinhibitors maybe one of potent chemotherapeutics.
These advances of indirubin in molecular biology and our previous studies of meisoindigo give a strong impetus to study the mechanism of meisoindigo in the treatment of CML and it antitumor activity and mechanism in solid tumor.
The mechanism of meisoindigo in treatment of CML
Meisoindigo effectively inhibited K562 cell and HL60 cell proliferation,and their IC_(50)s are 1.43μmol/L and 5.6μmol/L,respectively.Meisoindigo arrested K562 cells and HL60 cells in S phase and induced these cell apoptosis.Meisoindigo made the DNA on nucleosomes fracture and the DNA ladder appeared at 25μmol/L.The expressions ofβ-catenin and c-myc genes in HL60 cells were decreased by meisoindigo,but not significantly changed in K562 cells.The expressions of p-GSK-3β(Ser9),β-catenin and c-myc proteins were decreased,the GSK-3β(Ser9) protein was increased.Meisoindigo decreasedβ-catenin and c-myc expressions in HL60 cells,but did not affect these gene expressions in K562 cells.
The apoptosis related p53 and Bax protein expressions were increased and bcl-2 protein expression was decreased by meisoindigo.Meisoindigo decreased bcl-2 gene expression in K562 cells and increased Bax gene expression in K562 cells,but not in HL60 cells.In the study we also found that CDK2,cyclinA and cyclinB protein expressions were decreased by meisoindigo.These results showed that meisoindigo may affect Wnt signal pathway by inhibiting GSK-3β(Ser9) protein phosphoration which decreased the protein activity.The balance of phosphoration of Ser9 and Tyr216 was destroyed.Theβ-catenin protein in cytoplasm was phosphorated by GSK-3βand then degradated by proteasomes.The transcriptions and expressions of the oncogene(ie. c-myc,cyclinD) were decreased.On the other side,GSK-3βactivated the p53 activation which induced p53 mitochondria dependent apoptosis.The p53 and p21 proteins may inhibit CDK2 activity which arrests K562 cells and HL60 cells in S phase.
The antitumor activity and mechanism of meisoindigo against HT-29 cells in vitro and in vivo
Meisoindigo not only inhibited K562 cell and HL60 cell proliferation,but also showed potent inhibition on other solid tumor cells including MCF-7,A549 and HT-29 cells.In solid tumor cells,the HT-29 cells showed the most sensitive to meisoindigo,the IC_(50) is 4.3μmol/L.HT-29 cells were arrested at G2/M phase.The DNA fracture ladder was found.The RT-PCR results showed that theβ-catenin,c-myc and bcl-2 gene expression levels decreased and the Bax gene expression level increased.The Western blot assay showed GSK-3β,p53 and p21 protein expressions increased,and GSK-3β, CDK2 protein expressions decreased.The antitumor activity of meisoindigo in HT-29 cell xenograft results showed that meisoindigo could inhibit the HT-29 xenograft tumor growth,and the inhibitory rate was 47.64%at the 100mg/kg by the tumor weight compared with the control group.The pathological results of the tumor exhibited that meisoindigo could decrease the number of HT-29 cell nuclear divisions in a dose dependent manner.The immunohistochemistry results showed that meisoindigo could decrease theβ-catenin,c-myc and p-GSK-3βprotein expression.The antitumor activities of meisoindigo against HT-29 cells in vitro and in vivo showed that meisoindigo could inhibit colorectal HT-29 growth.The mechanism of meisoindigo against HT-29 may be to inhibit by GSK-3β(ser9) phosphorylation and CDK2.GSK-3 activity was negatively regulated by phosphorylation on Ser9 and positively regulated by phosphorylation on Tyr 216.The meisoindigo destroied the balance between GSK-3β(Ser9) phosphorylation and GSK-3β(Tyr216) phosphorylation.Thus,the activity of GSK-3βrelatively increased.On one side,theβ-catenin protein was phosphorated by GSK-3βprotein and degradated by proteasomes.The transcription and expression of the oncogenes(ie.c-myc,cyclinD) decreased.On the other side,GSK-3βactivated the p53 activation which induced p53 mitochondria dependent apoptosis.Another side,meisoindigo may directly and indirectly inhibit CDK2 activity by p21 protein which is an endogenous CDK inhibitor.Because the GSK-3βactivity increased which resulted in p53 and p21 protein expression increase.
In summary,the mechanism of meisoindigo in the treatment of CML may be related to its inhibition GSK-3β(Ser9) and CDK2 activities.The studies showed that meisoindigo potently inhibited colorectal cancer HT-29 cells in vitro and in vivo.The mechanisms of meisoindigo against HT-29 cells may be similar to it against CML.The results in HT-29 cells showed that meisoindigo may be applied for treatment of patients with colorectal cancers in the future.
目前,大量研究结果表明,靛玉红类化合物对糖原合成酶激酶GSK-3β(glycogen-synthase kinase,GSK)和细胞周期素依赖激酶CDK(cyclin-dependentkinase,CDK)有抑制作用,GSK-3β和CDKs都是丝/苏氨酸蛋白激酶,二者与丝裂原激活激酶(mitogen-activated kinase)及CDK样激酶(CDK-like kinase)同属于CMGC(Cyclin-dependent kinase,Map kinase,Glycogen synthase kinase and Cdk likekinases,CMGC)蛋白家族。GSK-3β是细胞内多种重要信号通路中重要的调控蛋白。GSK-3β是一种多功能的蛋白激酶,不仅参与细胞内的糖代谢,而且与细胞的分化、增殖和凋亡有密切关系,参与细胞内胰岛素信号通路、Wnt信号通路和NF-κB信号通路等。GSK-3β与人类糖尿病、神经退行性疾病、双向情感障碍和肿瘤等疾病的发生有密切的关系。肿瘤细胞一个重要特征是细胞周期调控机制的破坏导致细胞无限制地增殖。CDK和细胞周期素(cyclin)是细胞周期重要的调控蛋白,对细胞周期的准确调控起着重要作用,细胞周期的准确调控对细胞的增殖、分化和凋亡十分重要。当细胞周期调节出现异常时,在人类会导致各种疾病的发生如肿瘤、神经退行性疾病、心血管疾病和病毒感染等。因此,GSK-3β和CDK已经成为目前抗肿瘤药物研究的重要靶点。
GSK-3β是Wnt信号通路中重要的调控蛋白,Wnt信号通路与肿瘤的发生有密切关系,特别是在结肠癌中发现Wnt信号通路中APC(adenomatous polyposis coli,APC)、β-粘连蛋白(β-catenin)等多种蛋白异常,目前认为Wnt信号通路调节异常是结肠癌的重要发病机制之一。因此,GSK-3β抑制剂可能是治疗结肠癌的重要潜在药物之一。
基于以前对甲异靛的作用研究和目前分子生物学对靛玉红类化合物的研究进展,我们对甲异靛治疗慢性髓性白血病的作用机制和对实体瘤的抗瘤活性及作用机制进行了研究。
甲异靛治疗慢性髓性白血病机制研究
甲异靛在体外对K562细胞和HL60细胞增殖有明显的抑制作用,其IC_(50)分别为1.43μmol/L和5.60μmol/L,甲异靛能使K562细胞和HL60细胞阻滞在S期,诱导细胞凋亡,核小体DNA断裂,出现典型的DNA ladder条带。甲异靛能使HL60细胞Wnt信号通路中β-catenin和c-myc基因表达下降,而对K562细胞中β-catenin和c-myc基因表达没有明显影响,甲异靛使Writ信号通路中p-GSK-3β(Ser9)、β-catenin和c-myc蛋白表达下降,GSK-3β(Ser9)蛋白表达增加。甲异靛能诱导K562细胞和HL60细胞凋亡相关蛋白p53和Bax蛋白表达增加,bcl-2蛋白表达下降。K562细胞和HL60细胞中CDK2、cyclinA、cyclinB蛋白表达减少。这些结果提示甲异靛可能是通过竞争性抑制GSK-3β(Ser 9)磷酸化,使细胞内GSK-3β(Ser9)和GSK-3βTyr216磷酸化的平衡破坏,GSK-3β活性相对增强。一方面GSK-3β活性相对增强后胞浆内的β-catenin磷酸化增加,胞浆内磷酸化β-catenin泛素化后被蛋白酶体降解,胞浆内磷酸化β-catenin含量下降,进入细胞核内β-catenin相对减少,因此,与细胞分化和增殖相关的基因和蛋白如c-myc和cyclinD1等的表达相应减少。另一方面GSK-3β活性增强后,GSK-3β能短暂地与p53结合并激活p53,通过p53线粒体凋亡途径,即p53-Bax线粒体凋亡途径。p53发生移位使线粒体内抗凋亡蛋白bcl-2和促凋亡蛋白Bax平衡破坏,导致细胞色素c释放,诱发细胞内促凋亡因子不可逆地释放而激发细胞凋亡。细胞周期调控的主要蛋白是CDKs和cyclins,CDK是细胞周期调节和RNA转录重要调控蛋白。甲异靛使K562细胞和HL60细胞CDK2蛋白和cyclinA蛋白表达减少,而p53蛋白和p21蛋白表达增加。这可能是甲异靛通过抑制CDK2蛋白活性,从而使K562细胞和HL60细胞在S期受到阻滞。
甲异靛对结肠癌HT-29抗肿瘤活性及机制研究
体外筛选结果表明,甲异靛在体外对乳腺癌细胞MCF-7、非小细胞肺癌A549和HT-29等多种实体瘤细胞增殖都有明显的抑制作用。在实体瘤中对HT-29细胞的抑制活性最强。甲异靛能抑制HT-29细胞生长和集落形成。使HT-29细胞阻滞在G2/M期,诱导细胞凋亡,细胞内核小体DNA断裂出现典型的梯形条带。在基因水平甲异靛能使HT29细胞内β-catenin、c-Myc和bcl-2基因表达降低,Bax基因表达增加。在蛋白水平甲异靛能抑制GSK-3β磷酸化,使Wnt信号通路中pGSK-3β(Ser9)、β-catenin和c-Myc蛋白表达下降,GSK-3β(Ser9)蛋白表达增加。甲异靛抑制周期素依赖激酶(CDK),使cyclinA、cyclinB、p53和p21蛋白表达增加,CDK2蛋白表达减少。甲异靛能使细胞内抗凋亡蛋白bcl-2表达减少,使促凋亡蛋白Bax表达增加。
在人HT-29细胞裸鼠异体移植瘤模型中,甲异靛对HT-29细胞裸鼠异体移植瘤生长有明显抑制作用,与空白对照组比较,甲异靛能较明显抑制裸鼠体内肿瘤的生长,肿瘤抑制率可达到47.60%。瘤组织病理学结果提示,甲异靛对人结肠癌HT-29裸鼠异体移植瘤的生长有一定的抑制作用,随着给药剂量的增加,癌细胞分裂相数有所减少,尤其是大剂量组更为明显。人HT-29细胞裸鼠异体移植瘤组织免疫组化结果表明,甲异靛能抑制裸鼠异体移植瘤组织内GSK-3β(ser9)磷酸化,GSK-3β的活性量增加,而p-GSK-3β蛋白、β-catenin蛋白和c-myc蛋白表达相应减少。
甲异靛对人结肠癌HT-29的体内和体外结果提示,对人结直肠癌HT-29抑制作用可能是通过抑制GSK-3β(Ser9)磷酸化和CDK2活性实现的。甲异靛抑制GSK-3β(Ser9)磷酸化后活性相对增强,胞浆中β-catenin磷酸化增加,胞浆中β-catenin磷酸化后被降解,因此,进入细胞核的β-catenin减少,抑制了细胞核内的c-myc等癌基因的转录和表达。GSK-3β活性相对增加还可引起p53线粒体凋亡途径激活,即GSK-3β-p53-bax途径诱导细胞凋亡。HT-29细胞阻滞在G2/M期,这提示甲异靛可能是通过抑制CDK2活性,CDK2活性降低可直接导致细胞阻滞在G2/M期。另一方面,甲异靛通过抑制GSK-3β使p53蛋白和p21蛋白活性增加,通过p52-p21-CDK2途径,进一步使HT-29细胞阻滞在G2/M期。
总之,甲异靛治疗慢性髓性白血病的机制可能是通过抑制GSK-3β(Ser 9)磷酸化,GSK-3β活性相对增强,一方面使Wnt信号通路中GSK-3β下游β-catenin被磷酸化后降解增加,进入细胞核内β-catenin减少,细胞核内与分化和增殖相关的基因和蛋白如c-myc和cyclinD1等的表达下降。另一方面GSK-3β-p53途径诱导细胞凋亡。甲异靛对实体瘤人结肠癌HT-29的抑制作用也可能是通过抑制GSK-3β(Ser9)磷酸化和CDK2活性实现的。甲异靛在体内和体外对HT-29细胞的抑制作用提示甲异靛在临床上对结肠癌可能有一定的治疗作用。
Meisoindigo,an indirubin derivative,plays an important role in the treatment of chronic myelogenous leukemia(CML).Indirubin is the active indigrent of Danggui Longhui Wan,a mixture of plants that is used in Traditional Chinese Medicine to treatment of CML,but indirubin shows poor solubility and absorption,and exhibites serious side action in gastricintestinal tract.In order to improve indirubin these characteristics,several analogues of indirubin were synthesized by the Institute Materia and Medica,Chinese Academy of Medical Sciences & Peking Union Medical College. One of these analogues,Meisoindigo,shows stronger antitumor activity and only minor toxicity.Meisoindigo was approved for treatment of CML by State Foods and Drugs Administration(SFDA),and Meisoindigo had taken place of indirubin in the treatment of CML in China.Previous studies showed that meisoindigo inhibited DNA and RNA synthesis in W256 cells,prevented microtubule aggregation,inhibited new vascular formation and induced cell apoptosis.Wu Yingli reported that meisoindigo had no effect on BCR/ABL activity and expression,which was the characteristic protein of CML.So it is valuable to study the mechanism of meisoindigo against CML further.
Recently,indirubin and its derivatives were reported to inhibit glycogen-synthase kinase-3(GSK-3) and cyclin-dependent kinase(CDKs) activitives.GSK-3 and CDKs are serine/threonine kinase.Both of them with mitogen-activated kinase and CDK-like kinase belong to CMGC superfamily.GSK-3 is an important regulatory protein in many signal pathways including insulin signal patyway,Wnt signal pathway and NF-κB signal pathway.Not only dose it take part in glycogen synthesis and metabolism,but also participates in cell differentiation,proliferation and apoptosis.The dysregulation of GSK-3βabnormalities associates with diabetes mellitus,Alzheimer's disease,bipolar mood disorder and cancer.CDKs and cyclins are important regulator proteins in cell cycle.The normal cell cycle regulation is a necessary for cell cycle from one phase to the next phase.Cell cycle is relative to cell proliferation and apoptosis.A characteristic of cancer cells is its cell cycle dysregulation,which results in cancer cell permanent proliferation.When cell cycle regulation becomes abnormalities,it will result in cancer, neurodegenerative disease and cardiovascular disease.GSK-3βand CDKs have become the targets of cancer therapy.
GSK-3βplays a central role in Wnt signal pathway which implicats in cell survive and diverse cancers.GSK-3βprotein overexpression has been found in human ovarian, colon and pancreatic carcinoma.In colorectal cancer,many protein mutations including APC,β-catenin were founded.The dysregulation of Wnt signal pathway is one of colon cancer etiopathogenisis.The GSK-3βinhibitors maybe one of potent chemotherapeutics.
These advances of indirubin in molecular biology and our previous studies of meisoindigo give a strong impetus to study the mechanism of meisoindigo in the treatment of CML and it antitumor activity and mechanism in solid tumor.
The mechanism of meisoindigo in treatment of CML
Meisoindigo effectively inhibited K562 cell and HL60 cell proliferation,and their IC_(50)s are 1.43μmol/L and 5.6μmol/L,respectively.Meisoindigo arrested K562 cells and HL60 cells in S phase and induced these cell apoptosis.Meisoindigo made the DNA on nucleosomes fracture and the DNA ladder appeared at 25μmol/L.The expressions ofβ-catenin and c-myc genes in HL60 cells were decreased by meisoindigo,but not significantly changed in K562 cells.The expressions of p-GSK-3β(Ser9),β-catenin and c-myc proteins were decreased,the GSK-3β(Ser9) protein was increased.Meisoindigo decreasedβ-catenin and c-myc expressions in HL60 cells,but did not affect these gene expressions in K562 cells.
The apoptosis related p53 and Bax protein expressions were increased and bcl-2 protein expression was decreased by meisoindigo.Meisoindigo decreased bcl-2 gene expression in K562 cells and increased Bax gene expression in K562 cells,but not in HL60 cells.In the study we also found that CDK2,cyclinA and cyclinB protein expressions were decreased by meisoindigo.These results showed that meisoindigo may affect Wnt signal pathway by inhibiting GSK-3β(Ser9) protein phosphoration which decreased the protein activity.The balance of phosphoration of Ser9 and Tyr216 was destroyed.Theβ-catenin protein in cytoplasm was phosphorated by GSK-3βand then degradated by proteasomes.The transcriptions and expressions of the oncogene(ie. c-myc,cyclinD) were decreased.On the other side,GSK-3βactivated the p53 activation which induced p53 mitochondria dependent apoptosis.The p53 and p21 proteins may inhibit CDK2 activity which arrests K562 cells and HL60 cells in S phase.
The antitumor activity and mechanism of meisoindigo against HT-29 cells in vitro and in vivo
Meisoindigo not only inhibited K562 cell and HL60 cell proliferation,but also showed potent inhibition on other solid tumor cells including MCF-7,A549 and HT-29 cells.In solid tumor cells,the HT-29 cells showed the most sensitive to meisoindigo,the IC_(50) is 4.3μmol/L.HT-29 cells were arrested at G2/M phase.The DNA fracture ladder was found.The RT-PCR results showed that theβ-catenin,c-myc and bcl-2 gene expression levels decreased and the Bax gene expression level increased.The Western blot assay showed GSK-3β,p53 and p21 protein expressions increased,and GSK-3β, CDK2 protein expressions decreased.The antitumor activity of meisoindigo in HT-29 cell xenograft results showed that meisoindigo could inhibit the HT-29 xenograft tumor growth,and the inhibitory rate was 47.64%at the 100mg/kg by the tumor weight compared with the control group.The pathological results of the tumor exhibited that meisoindigo could decrease the number of HT-29 cell nuclear divisions in a dose dependent manner.The immunohistochemistry results showed that meisoindigo could decrease theβ-catenin,c-myc and p-GSK-3βprotein expression.The antitumor activities of meisoindigo against HT-29 cells in vitro and in vivo showed that meisoindigo could inhibit colorectal HT-29 growth.The mechanism of meisoindigo against HT-29 may be to inhibit by GSK-3β(ser9) phosphorylation and CDK2.GSK-3 activity was negatively regulated by phosphorylation on Ser9 and positively regulated by phosphorylation on Tyr 216.The meisoindigo destroied the balance between GSK-3β(Ser9) phosphorylation and GSK-3β(Tyr216) phosphorylation.Thus,the activity of GSK-3βrelatively increased.On one side,theβ-catenin protein was phosphorated by GSK-3βprotein and degradated by proteasomes.The transcription and expression of the oncogenes(ie.c-myc,cyclinD) decreased.On the other side,GSK-3βactivated the p53 activation which induced p53 mitochondria dependent apoptosis.Another side,meisoindigo may directly and indirectly inhibit CDK2 activity by p21 protein which is an endogenous CDK inhibitor.Because the GSK-3βactivity increased which resulted in p53 and p21 protein expression increase.
In summary,the mechanism of meisoindigo in the treatment of CML may be related to its inhibition GSK-3β(Ser9) and CDK2 activities.The studies showed that meisoindigo potently inhibited colorectal cancer HT-29 cells in vitro and in vivo.The mechanisms of meisoindigo against HT-29 cells may be similar to it against CML.The results in HT-29 cells showed that meisoindigo may be applied for treatment of patients with colorectal cancers in the future.
引文
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