Exploration Of Folate Receptor Mediated Antitumor Activity And Mechanism For Folate-macromolecular Conjugates

Folates are low molecular weight pterin-based vitamins involved in one-carbontransfer reactions during the synthesis of nucleic acids and metabolism of amino acids,and the form of folate added to fortified foods and vitamin supplements is folic acid.Because humans and other mammal cells lack key enzymes of the folate biosyntheticpathway, they cannot synthesize folate, and must obtain it from exogenous sources forsurvival and proliferation.There are two mechanisms for the cellular internalization of the vitamin. The firstmechanism involves a low-affinity (Kd~5×10-6mol/L) membrane-spanning proteinthat transports reduced folates directly into the cell cytosol. The second mechanismuses a high affinity (Kd~10-10mol/L) glycosylphosphatidylinositol-anchoredglycoprotein receptor, generally referred to as the folate receptor, which preferentiallymediates the uptake of oxidized forms of folate (e.g. folic acid) into the cell byendocytosis. The folate transporter is present on virtually all cells, whereas thehigh-affinity folate receptor is expressed at high levels mainly on cancer cells.Overexpression of folate receptor by cancer cells might provide the cells with agrowth advantage relative to neighbouring normal tissue, and highly undifferentiatedmetastatic cancers express considerably more folate receptor than their localized,low-grade counterparts.The discovery of high-levels expression of folate receptor on many human cancercells has rendered the folate receptor an attractive candidate for development of tumorspecific therapeutics. In this role, the folate receptor can effectively serve either oftwo distinct functions: one as a vehicle for the non-destructive trafficking ofextracellular diagnostic agents into the cytoplasm of targeted tumor cells, and theother as a simple tumor marker that allows ligand-mediated enrichment of therapeuticmacromolecules including toxins, polymers, gene therapy vectors, andliposome-encapsulating drugs on tumor cell surfaces.The main object for this study is also folate receptors overexpressed in tumor cells, 4英 文 摘 要and to evaluate the antitumor activity and mechanism of some folate- macromolecules(synthesized by our lab ) by the point of view of tissue, cellular and molecular level.The main molecule used in the study was folate-dextran conjugate which was whichhas been authorized a patent by State Intellectual Property office of P. R. China.Thefolate –macromolecules including folate-carboxymethylized dextran conjugates andfolate-PEG conjugate.The first step for the study was to determine folate receptor expression in 18kinds ofcell lines like KB,HeLa229,HeLa,SGC-7901,SMMC7721,Bel7402,SPC-A-1,SK-BR-3,Raji,CNE2, L02;SCI-375,SK-OV-3 ,MCF 7 by determine itsmaximum binding sites and affinity by performing the classic radioligand bindinganalysis. Our results show that KB,HeLa229,HeLa,SGC-7901,SMMC7721,Bel7402,SPC-A-1,overexpresses folate receptors and its radioligand bindingbehavior was shown to be of high affinity, satuarable competitable and reversible. The125I-folate binding sites on the surface of these tumor cells were all> 60fmol/106cell-1;The KD values of folate receptors of different cells were all<5.06×10-10mol/l. Thetumor cells expressed more folate receptors than those normal hepatic cells, such asL02. Up to now, there isn't any report about folate receptor overexpressed on cells ofSMMC7721, Bel7402 and SPC-A-1. The results obtained in our study expanded thetumor cell lineage which overexpress folate receptors and these cells can be used asnew targets for non-destructive diagnosis and tumor-specific drug and gene delivery.Folate receptor bidnding studies indicated that folate-dextran conjugate,folate-carboxymethylized dextran conjugates and folate-PEG conjugate.couldspecifically binding to folate receptor bearing tumor cells which described above, andcould competitivel...