The Antitumor Activities And Mechanisms Of Meisoindigo Against Chronic Myelogenous Leukemia And Human Colorectal Adenocarcinoma HT-29

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 CMLMeisoindigo effectively inhibited K562 cell and HL60 cell proliferation,and their IC₅₀s 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 IC₅₀ 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.