Affinity Mechanism Of Recombined Toxin LHRH-PE40 With LHRH Receptors On The Membrane Of Target Cells And Apoptosis Study Of Target Cells Induced By LHRH-PE40

The targeted therapy on tumor is one of the hottest topics nowadays inthe field of medicine and biology, which see some special molecular on thesurface of tumor cells as targeting site and attack them locally with a littleor no effect on normal cells. Recombined toxin LHRH-PE40 (luteinizinghormone releasing hormone-pseudomonas aeruginosa exotoxin 40), one ofthe new targeting drugs against cancer, is a kind of fusion proteins. Itsgenes including LHRH gene and PE40 gene were recombined byengineering way and expressed in E.coli. The principle is specific bindingby its LHRH part with LHRH receptor on the surface of targeting cell andthen transport PE40 by endocytosis to kill target cell specially. In this studytargeting cells are some tumor cells, which express LHRH receptors muchhigher than the other normal cells. One of our aims is for detecting thelethal effect of new targeting drug against cancer specially. The other aim isto detect its high efficiency. It is that a little amount of LHRH-PE40 couldinduce targeting cells apoptosis. Our study connected with not only clinicalpractice but also patient's life. So it seems greatly significant. BecauseLHRH-PE40 brings new ideas itself, the detection and conclusion about itare all the first reports.As the targeting part of LHRH-PE40, LHRH can recognize and bindLHRH receptors specially, because LHRH receptors distribute differentlybetween normal cells and cancer cells. It was report that the binding on themembrane of target cells is much more than on that of counterpart normalcells. One of the methods in this study is ELISA, in which OD valuerepresents the binding ability of LHRH receptors with LHRH-PE40. theresult was analyzed by the SPSS10.0 Statistic Software. The OD valueindirectly showed the distribution of LHRH receptors on membrane ofdifferent cells. The cells in vitro such as Hela cells, melanoma cells (A375)were detected for their affinity, saturability, and time relation of LHRHreceptors binding with LHRH-PE40. Results showed that the quantity ofLHRH receptors on the surface of Hela cells is relatively higher than thaton A375. After LHRH-PE40 competed LHRH receptors on the surface ofHela cells with LHRH and TSH (throid stimulating hormone) respectively,the OD value of this two group was analysed by SPSS10.0 software(P<0.01). So it is greatly significant to the binding of LHRH-PE40 withLHRH receptors on the surface of target cells. As the bullet part of LHRH-PE40, PEA is a toxogen of LHRH-PE40.It is composed of three domains ( Ⅰa, Ⅰb, Ⅱand Ⅲ). They holdrespectively these functions of recognizing and binding receptors,trarsmembrane and the activity of ADP transribosylase. The cytotoxicity ofPEA is by catalyzing elongation factor 2 ribosylated and inhibiting proteinsynthesis. Its merit is special structure, strong toxicity, easy internalization,and high speciality. PE40 is the mutant part of Ⅰa deleted from PEA. AfterPE40 gene and LHRH gene were recombined, the expressive proteinpossesses new recognizing and binding function, which can killcorresponding targeting cells specially. Under light microscope(LM),LHRH-PE40 toxicity assay showed that Hela cells and A375 wereeffectively killed but no effect on Hut102 and original chick embryofibroblast, because the affinity of LHRH-PE40 with LHRH receptors on themembrane of Hela cells and A375 is much higher than that of Hut102 andoriginal chick embryo fibroblast. Morphologic observation identified withthe results of ELISA assay. The binding of LHRH-PE40 with its receptorson the surface of cells holds high specialty and saturability. Only afterLHRH-PE40 mediated by LHRH receptor enters into cells, its toxicity canfunction to cells, which express LHRH receptors positively. One of LHERH-PE40 characteristics is that it binds specially withLHRH receptors, which is also targeting marker of recombined toxinLHRH-PE40. Spiegelmers were adopted to detect this feature. Spiegelmersare mirror-image, high-affinity L-enantiomeric oligonucleotide ligandscomposed of L-2'-deoxyribose units. Spiegelmer specifically binds tonatural GnRH with high affinity and blocks its functional activity. ThisSpecial and targeting Spiegelmers were mixed with LHRH-PE40, and thenthe combining solution was added into the solution of targeting cells.Experimental control group was treated without Spiegelmers. The resultswere observed under LM (light microscopy, ×100) that Spiegelmersinhibited the binding of LHRH-PE40 with LHRH receptors on membraneof Hela cells and A375. That is to say that Spiegelmers inhibit the toxicityof LHRH-PE40, while the cytotoxicity of LHRH-PE40 on control group isvery clear. This experiment showed that LHRH part of LHRH-PE40 holdsthe characteristic of nature LHRH binding LHRH receptors. The indirectcompetition-inhibition experiment is a best supplement to above LHRHreceptor assay. The other method in this study is the detection of LHRH-PE40inducing target cells apoptosis. The apoptotic body and chromaticcollection to nuclear margin were observed by transmission electronmicroscope (TEM). And also apoptotic signs were also showed under SEM(scan EM). Light straps of ladder-shaped were detected in 50bp, 200bp,...