Kinase Signaling And Epigenetic Regulation By Bortezomib And Bortezomib Delivery System In Leukemogenesis

Cell signaling transduction pathway plays an important role in tumor initiation.The essence of cancer pathogenesis is the aberrant cell signal transduction.To investigate an anti-cancer drug for the critical target in cell signal pathway,it is of great significance for accurate cancer therapy to study the molecular mechanism underlying in cancer pathogenesis,aberrant epigenetics and sustained activation of protein kinase,look for innovative therapeutic drug,and construct novel drug delivery system.1.Bortezomib inhibits chronic myeloid leukemia growth through kinase and DNA methylation crosstalkIt is reported that,in CML,DNMTs are highly expressed and aberrant DNA methylation is associated with worse outcomes,but the mechanisms underlying this phenomena remains poorly understood,and further effort should be put into getting a better CML therapeutic outcome.Here bortezomib was employed for study in vitro andin vivo.We demonstrated BCR/ABL kinase as a positive modulator for DNA methylation machinery.In agreement with this,the expression of ABL kinase was positively correlated with the levels of DNMTs in CML patients.BCR/ABL overexpression upregulated DNMT leading to DNA hypermethylation;in contrast,small interference RNA mediated-depletion or pharmacological inhibition of BCR/ABL suppressed DNMT expression,reduced global DNA methylation and restored the expression of tumor suppressors CDHI via promoter hypomethylation.Mechanistically,BCR/ABL-driven DNA hypermethylation occurs through NFκB signaling,since BCR/ABL dysfunction impaired NFκB pathway.Ectopic NFκB expression increased,whereas NFrκB inhibition decreased,DNMT transcription leading to the in-parallel change of DNA methylation.Functionally,blockade of BCR/ABL inhibited CML cell clonogenic potential in vitro.Notably,the expression of DNMTs,the phosphorylation of NFrκB and the levels of DNA methylation in tumors were significantly abolished.Altogether,our findings support a novel paradigm in CML leukemogenesis in which constitutively active BCR/ABL kinase drives leukemia growth through NFκB-DNMT axis.In another aspect,it is conformed that bortezomib inhibits DNA hypermethylation,while it can also inhibit the expression of Bcr/Abl kinase and transcription factor Spl.Given that miR-29b inhibits Spl,Spl transactivates DNMT1,and Spl activates Bcr/Abl,thus we conclude the relationship among transcription factor Spl,kinase Bcr/Abl and DNA methylation in CML.Bortezomib and miR-29b inhibit not only aberrant tyrosine kinase,but also DNA hypermethylation.It is elucidated though xenograft mice model,that leukemia tumor with combination treatment of bortezomib and miR-29b grows lowest,so that we conclude bortezomib and miR-29b combination is more potent for leukemia therapy.2.AuNP/HDL/Bortezomib nanoparticle conjugates as molecularly targeted therapy for leukemiaWhile the major leukemia patients achieve remission with upfront chemotherapy,most patients relapse or become refractory to current therapies.Thus,innovative therapeutics that challenges existing paradigms is desirable.Due in part to their biocompatibility and tailorability,gold nanoparticle(AuNP)conjugates hold therapeutics promise.High density lipoprotein(HDL)has been implicated in cholesterol delivery in some malignancies.While HDL-AuNP has been utilized to deliver nucleic acid,HDL-AuNP-delivered chemo-compounds remain unexplored.Here,we report a gold nanoparticle-based therapeutic system(AuNPs-HDL-Bortezomib)to leukemia by delivering bortezomib,an FDA approved proteasome inhibitor and widely used in clinic.Here,we report a gold nanoparticle-based delivery of leukemia therapeutics HDL-AuNP-Bortezomib are synthesized using a 20 nm gold nanoparticle template to control conjugate size and ensure a spherical shape to engineer HDL-like nanoparticle containing bortezomib.The size and shape of the HDL-AuNP measured by TEM showed that the nanoparticles are electrostatically stable and 25 nm.The encapsulation efficiency of AuNP-HDL-Bortezomib is about 90%.AuNP-HDL-Bortezomib promotes leukemia cell apoptosis in vitro and ex vivo,inhibits Sp1,Axl and DNMT1 expression,and reduces global DNA methylation.Importantly,systemically delivered HDL-Au-NP-Bortezomib conjugates in leukemia-bearing mice reduced tumor burden and progression without obvious side effects.Together,these findings highlight HDL-AuNP-mediated Bortezomib delivery as an unconventional therapeutic strategy in leukemia.3.A nucleolin-DNMTl regulatory axis in acute myeloid leukemogenesisNucleolin overexpression and DNA hypermethylation have been implicated in cancer pathogenesis,but whether and how these aberrations cooperate in controlling leukemia cell fate remains elusive.In this report,we provide the first mechanistic insights into the role of nucleolin in leukemogenesis through creating a DNA hypermethylation profile in leukemia cells.We found that,in leukemia patients,nucleolin levels are significantly elevated and nucleolin overexpression strongly associates with DNMT upregulation and the shorter survival.Ectopic nucleolin expression augmented leukemia cell proliferation,whereas nucleolin dysfunction by RNA interference and inhibitory molecule AS 1411 led to the blockade of leukemia cell clonogenic potential in vitro and the impairment of tumorigenesis in vivo.Mechanistic investigations showed that nucleolin directly activates NFκB signaling,and NFκB activates its downstream effector,DNA methylation machinery.In agreement with this,enforced nucleolin expression enhanced NFκB phosphorylation and upregulated DNMT1 that is followed by an increase of DNA methylation;by contrast,nucleolin inactivation by AS 1411 or siRNA dephosphorylated NFκB and abrogated DNMT1 expression,which resulted in a reduction of global DNA methylation,restored p15INK4B gene expression and DNA hypomethylation on p15INK4B promoter.Of note,NFκB inactivation decreased,whereas NFκB overexpression increased DNMT1 promoter activity and endogenous DNMT1 expression.Collectively,our studies identify nucleolin as an unconventional epigenetic regulator in leukemia cells and demonstrate nucleolin-NFκB-DNMT1 axis as a new molecular pathway underlying AML leukemogenesis,providing potentially a new target for therapeutic intervention.4.Design and construction for Gold nanoparticles-based drug nano-carrierAccording to the relationship study between nucleolin and DNA methylation,we demonstrate the NCL-NFκB-DNMT1 controls leukemia growth.Given that nucleolin is highly expressed in cancer cell membrane and it is the target for its aptamer AS1411,AS1411 become the recognition molecule for cancer cell,thus it makes the nano-carrier functionalized for cancer therapy.This is of significance for designing and constructing nano-carrier for drug delivery.