Human Apolipoprotein(a) Carboxyl-terminal Kringles' Influence On Angiogenesis & Tumor Growth

Angiogenesis plays an important role in tumor growth and progression,so inhibition of tumor angiogenesis has been a major methods in tumor treatment.It is well studied that angiostatin is an effective tumor angiogenesis inhibitor,which is naturally derived from plasminogen(PLG).Angiostatin contains the former four kringles,from kringle 1 to kringle 4,of PLG.Further study about PLG's kringles revealed that kringle 5 did inhibit angiogenesis well.Human apolipoprotein(a)[Apo(a)]is the characteristic apolipoprotein of lipoprotein(a) [Lp(a)].Apo(a) is composed of several kringles that share high homology with kringle 4 and 5 respectively.We studied kringleⅣtype 10 to kringleⅤof Apo(a),the most homologous kringles between Apo(a) and PLG,in detail as our major.RHAKs(RHAKA, RHAKB and RHAKC) composed of different kringles from kringleⅣtype 9 to kringleⅤwere expressed by Pichia pastoris firstly.And individual anti-angiogenic ability was illustrated through the structure and feature characterization and several angiogenesis protocols.Further more,RHAKB,composed of kringleⅣtype 10- kringleⅤ,was expressed by human colorectal carcinoma cells(HCT 116 cells).The influence of RHAKB's expression on both tumor cell proliferation in vitro or in vivo and tumor growth were examined in order to clarify its inhibitory effect on angiogenesis-dependant tumor growth,and its future in tumor angiogenesis gene therapy.Human Apo(a) cDNA was reversely transcripted from human liver tissue.Upon the high repetition and homology ofApo(a) kringles,69L and PL,containing 10688 to 12355 and 12301 to 13880 of Apo(a) cDNA respectively,were cloned into pMD18-T vector.Moreover,three RHAKs,with RHAKA for kringleⅤ,RHAKB for kringleⅣtype 10-kringleⅤand RHAKC for kringleⅣtype 9-10-kringleⅤ,were cloned into pPICZαA.After the successful transformation of three recombinant vectors into Pichia pastoris X-33, we screened each best strain in recombinant expression through Mut phenotype determination and SDS-PAGE.RttAKs were expressed in large Scale under the induction of methanol.RHAKs were recovered through the precipitation in saturated ammonium sulfate.The precipitates were re-dissolved and dialyzed before the purification through His·Bind(?) chromatography.After the primary purification,RHAKs were further analyzed in reverse-phase high performance liquid chromatography.The N-terminal six amino acid residues of RHAKB were sequenced,as suggested that the secretory signal peptideα-factor was cleaved during the secretory expression.Glycosylation prediction showed that there were several glycosylated sites,both N-linked and O-linked,distributed in RHAKs.The carbohydrates of RHAKA and RHAKB were stained and validated through periodic acid - Schiff(PAS) reagent.Thiol and sulfide quantitation indicated no free thiols existed in RHAKs.Moreover,no polymers but monomers were found in reduced SDS-PAGE.Both glycosylation and disulfide bonds formation were major points of kringle structure,as could be maintained well when being secreted by Pichia pastoris.Furthermore,RHAKs anti-angiogenic abilities were examined through endothelial proliferation inhibitory assay,wound migration assay and chick chorioallantoic membrane(CAM) assay.All the RHAKs inhibited the proliferation of human umbilical vein endothelial cells(HUVEC) and neo-angiogenesis on CAM as well. What's more,RHAKB inhibited HUVEC migration induced by bFGF.However,neither RHAKA nor RHAKB could affect the proliferation of HCT 116 cells.To accomplish the expression of RHAKB in HCT 116 cells with the help of pcDNA^TM 3.1/myc-His(-) A,we designed to append the Apo(a) signal peptide sequence to the 5' end of RHAKB after two step PCR.Signal peptide prediction showed that there were several cleavage sites located between the Apo(a) signal peptide and RHAKB.Targetting of RHAKB was predicted to be secreted outside the cell.The examination of transiently transcripted HCT 116 cells through immunofluorescence microscopy revealed that RHAKB was expressed in a secretory way.The transient transcription of pcDNA3.1(-) A-RHAKB into HCT 116 cells didn't inhibit the proliferation of themselves.Stably transfected HCT 116 cell strains were screened out through G 418 resistance.Both RHAKB mRNA and secreted protein of all screened strains were tested through RT-PCR and Western Blots.To establish some animal tumor models,the normal HCT 116 cells and the cells stably transfected with pcDNA3.1(-) A or pcDNA3.1(-) A-RHAKB were inoculated into BALB/c nude mice subcutaneously.After dynamic measurement of subcutaneous tumor nodules,we found that RHAKB stable expression HCT 116 cells held slower growth rates than the others.Thirty days after inoculation,both normal HCT 116 cells(eleven times) and those stably transfected with pcDNA3.1(-) A(five times) formed larger tumor nodules than the rest ones.Pathological diagnosis approved that each nodule was composed of the inoculated adenocarcinoma cells.Immunohistochemistry of human proliferative cell nuclear antigens(PCNA) indicated that no difference existed among the proliferative tumor cells of all tumor nodules.Through the detail study of kringleⅣtype 10 to kringleⅤof Apo(a) and some other related kringles,we drew out the following conclusions.1.Pichia pastoris,as a widely used expression host,could be used for recombinant kringles,since it can maintain some important kringle features.2.All three RHAKs inhibit angiogenesis well,but almost no difference existed among them,as may indicate a key status of kringleⅤof Apo(a) in anti-angiogenesis.3.Stable expression of RHAKB inhibit the formation and progression of tumor nodules, as was achieved without affecting the proliferation of tumor cells.All above results indicated that kringleⅣtype 10 to kringleⅤof Apo(a) inhibit tumor growth mainly through anti-angiogenesis,as provided future study in natural tumor antagonist and related gene therapy with meaningful evidences.