Preliminary Study On The Synthesis And Anti-tumor Effect In Vitro Of Methotrexate Modified By The Peptide

Objective To synthesize methotrexate modified by LH-RH peptide (LH-RH-MTX) and study preliminarily on its anti-tumor effect in vitro. Methods LH-RH - MTX was synthesized by coupling APA covalently to [D-Lys6]LH-RH. Its structure was identified by ESI-MS, IR and 1H-NMR. MCF-7 human breast carcinoma cell line highly expressing LH-RH receptors and K562 human erythroleukemia cell Iine lowly expressing LH-RH receptors were served as the tested cells. The cell proliferation inhibition rates of LH-RH-MTX were detected by MTT colorimetric assay. The effect of LH-RH-MTX on the cell cycle and apoptosis rates were detected by flow cytometry. The effect of LH-RH-MTX on the expression of LHRHR was determined by immunocytochemistry assay and the expression of LHRHR mRNA was determined by RT-PCR technique.Results LH-RH-MTX was flaxen crystalline powder with the yield of 64.98% and melting point of 165~166oC. The molecular weight of LH-RH-MTX was 1268.57, which coincided with the theoretical value. The inhibition rate of LH-RH-MTX on MCF-7 cells was much higher than that on K562 cells, and the inhibition rate of LH-RH-MTX on MCF-7 cells was much higher than that of free MTX at the same concentration. The inhibition rate of LH-RH-MTX on rat bone marrow mononuclear cells was less than that of free MTX. The number of MCF-7 cells in G0/G1 phase significantly decreased and in S phase increased after administration of LH-RH-MTX. The apoptosis rate of LH-RH-MTX group significantly increased compared with the control group and MTX group. In contrast to the control group and MTX group, the positive rate of LH-RH-MTX group significantly decreased by immunocytochemistry assay. RT-PCR results showed that the relative expression of LHRHR mRNA of LH-RH-MTX group markedly decreased compared with the control group and MTX group.Conclusions The inhibitory action of MTX modified by LH-RH peptide significantly increased on MCF-7 cells and decreased on rat bone marrow mononuclear cells. LH-RH-MTX had an influence on the cell cycle progression of MCF-7 cells, playing the role in S phase. LH-RH-MTX could induce apoptosis of MCF-7 cells and significantly down regulate the expression of LHRHR. The anti-tumor drug can be delivered to targeting tumor cells expressing LHRHR with the LH-RH-oriented elements, which plays an anti-tumor role with LH-RH binding to its receptor specifically. It is realizable to reduce drug side effects, increase the therapeutic index and achieve tumor-targeted therapy.