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40000350003000025000200001500010000500000.16 0.8 4 20 100Concentration (µM)Dau·HCl[D-Lys6(Dau=Aoa)]GnRH-II[D-Lys6(Dau=Aoa-GFLG)]GnRH-II[D-Lys6(Dau=Aoa)]GnRH-I[D-Lys6(Dau=Aoa-GFLG)]GnRH-IMCF-7HT-29Fluorescence intensity100009000800070006000500040003000200010000Fluorescence intensity1 2 3 4 5Concentration (µM)[D-Lys6(Dau=Aoa)]GnRH-II[D-Lys6(Dau=Aoa-GFLG)]GnRH-II[D-Lys6(Dau=Aoa)]GnRH-I[D-Lys6(Dau=Aoa-GFLG)]GnRH-IFluorescence intensity100009000800070006000500040003000200010000Fluorescence intensity7000060000500004000030000200001000000.16 0.8 4 20 100Concentration (µM)Dau SzI-2901 SzI-2911 SzI-2903 SzI-29131 2 3 4 5Concentration (µM)SzI-2901 SzI-2911 SzI-2903 SzI-2913Fig. 2. Cellular uptake of the daunomycin and the GnRH conjugates.In the case of both cell cultures the most effective compound was the free daunomycin. Theresults might be explained by the different mechanism of cellular uptake of the compounds.GnRH conjugates can be taken up by receptor mediated endocytosis, while daunomycinmay enter the cell by passive diffusion. In Figure 2, fluorescence values are illustratedwhich are proportional with cellular uptake of the conjugates.New GnRH-based drugTable 1. Cytostatic and cytotoxic effect of thedaunomycin and GnRH conjugatesDaunomycin Hydrochloride[D-Lys 6 (Dau=Aoa)]GnRH-II[D-Lys 6 (Dau=Aoa-GFLG)]GnRH-II[D-Lys 6 (Dau=Aoa)]GnRH-I[D-Lys 6 (Dau=Aoa-GFLG)]GnRH-IGnRH-III(Dau=Aoa)GnRH-III(Dau=Aoa-GFLG)*Only one data pointCytostasisIC 50 (µM)MCF-7HT-290.1-0.34.27±0.056.29*4.1±0.4022.4±6.94.35±1.974.53±1.5419.27±3.1019.22±2.3720.21*24.2±4.705.25±0.0715.90±5.50CytotoxicityIC 50 (µM)HepG2non toxicnon toxiccontaining conjugates weresynthesized. These conjugatesconsist of daunomycin andGnRH-II molecules which arecoupled to each other directly orvia Cathepsin B labile spacer(GFLG). Cytostatic effect andcellular uptake of conjugates weredetermined on two differentcancer cell cultures (MCF-7 andHT-29). Cytotoxicity of GnRH-IIconjugates was also studied onHepG2, human hepatocellularcarcinoma. Cytostatic effect ofthe conjugates was cell typedependent; [D-Lys 6 (Dau=Aoa)]-GnRH-II had higher (IC 50 : 4.27 µM) cytostatic activity on MCF-7 cells than the one withspacer (IC 50 : 6.29 µM). This difference was not observed on HT-29 cells (IC 50 : 19.29 and19.22 µM). Cytostatic effect of the GnRH-II conjugates was similar than it was shown bythe appropriate GnRH-I or GnRH-III conjugates. None of the GnRH-II conjugate wascytotoxic on HepG2 cell culture.Conjugates can be internalized in a concentration dependent manner on MCF-7 andHT-29 cells. In the case both cell culture [D-Lys 6 (Dau=Aoa-GFLG)]GnRH-II can be takenup more effectively than the [D-Lys 6 (Dau=Aoa)]GnRH-II. No significant difference wasobserved when we compared the internalization ability of GnRH-I and GnRH-IIconjugates. These conjugates will be potential candidate in the targeted tumor therapy.AcknowledgementThis work was supported by grants from the Hungarian National Science Fund (OTKA NK 77485, F67884, K 81596) and GVOP-3.2.1.-2004-04-0005/3.toxicReferences1. White, R., et al. PNAS 95, 305-309 (1998).2. Millar, R.P. Trends in Endocrinology and Metabolism 14, 35-43 (2003).3. Joseffson, E., et al. Immunology 88, 140-146 (1996).4. Krontic, S., et al. Peptides 21, 1941-1964 (2000).5. Günthert, A., et al. Eur. J. Endocrinol.153, 613-625 (2005).6. Neil, J., et al. Trends Endorinol. Metab. 15, 383-392 (2004).7. Gründker, C., et al. Eur. J. Endorinol. 151, 141-149 (2004).8. Szabó, I., et al. Bioconj. Chem. 20, 656-665 (2009).1-5523