| Antiproliferative Effect and Mechanismof Nolatrexed DihydrochlorideABSTRACTOBJECTIVE Nolatrexed dihydrochloride , a novel "nonclassical"thymidylate synthase (TS) inhibitor was designed based on the threedimensional structure of the active site of TS determined by X-ray crystallography. The aims of this research about the drug were as follows:(1). To observe the antiproliferative effect of nolatrexed dihydrochloride in vitro on 10 nonselected cancer cell lines and 3 normal cells in order to select the sensitive cancer cell lines.(2). To evaluate the therapeutic effect of nolatrexed dihydrochioride on mice bearing SRS-82 and s-i 80 ascitic type carcinomas or solid tumors in vivo. (3). To analyse the distribution characteristics of nolatrexed dihydrochloride in the organs and tumor tissues of mice bearing solid tumor .(4).To discover the mechanism of nolatrexed dihydrochloride in the view of apoptosis, and to find out the relationship between the drug concentration and the cell ratio of apoptosis. (5).To find out the influence of different concentration nolatrexed dihydrochloride on the level of thymidylate synthase messenger RNA of SRS-82 cancer cell line by reverse transcript polymerase chain reaction (RT-PCR) with the mouse TS cDNA primers designed by us, to see if there was cascade reaction in inhibiting the activity of thymidylate synthase by the drug.METHODS (1).Antiproliferative effect of the drug on cultured cells in vitro were assayed by MTT method.(2).Anticancer effect of the drug on mice bearing ascitic type carcinoma were evaluated by increasing the life span after a treatment schedule of 7 days with a dosage of lOOmg.kgt.d1?75mg.kg-1 and 5Omg.kg-1d-1 respectively; The inhibitory effect of-5-nolatrexed dihydrochloride on mice bearing solid tUInors were evaluated bydecreasing the weight of the tumor tissues. The difference among multi-grouPs mean value was analysed by ANOVA and the difference betWeen twogrouPs was analysed by t-test. (3).The concentration of nolatrexeddihydrochloride in the tissues of the organs and tumor were assayed by highperformance liquid chromatograPhy (HPLC) at 257nm. (4). Morphologicalchanges of typical aPoptosis were observed by transmission electronmicroscopy and ordinary microscopy; DNA fragment was assayed byagarose gel electrophoresis; Cell ratio of aPoptosis were measured by flowcytometry. (5). The influence of nolatrexed dihydrochloride on the TSmRNA level of SRS-82 cell line was measured by RT-PCR.RESULTS (l).The 50 percent inhibitory concentration of the drug fOrcancer cell line K562, NS-l, HepG2, MCF7, C6, LoVo, QSG7703, SRS-82,T24and S-l80 were 55.8, 29.1, 21 .3, 16.9, 57.5, 0.012, 95.2, 10.2, 0.l9, 8l .3and 3.25uM respectively; The IC50 for normal cell HEK293, CHO and Fbwere 57.5, 187.6 and 206.5uM respectively. (2).The life span of micebearing ascitic type carcinoma treated with nolatrexed dihydrochloride wereincreased significantly; The weight of taor tissues in mice treated withnolatrexed dihydrochloride were decreased significanly, the theraPeuticeffect of nolatrexed dihydrochloride was obvious in vivo. (3).The peakconcentration of the drug in blood, heart, liver, spleen, lung, kidney, brainand tumor tissues were l2.8, 15.7, 40.l, 1 l .5, 17.7, 58.4, l6. l and 1 .5ug.ml-lrespectively. (4).It was confirmed by morphology and agarose gelelectrophoresis that causing aPoptosis is one of the aniproliferativemechedsm fOr nolatrexed. Flow cytometry showed that the maxiumpercentage of apoptosis was appeared at 480 min after the cells were exposedto the drug and cell aPoptosis at 0, 3.l2, 6.25, l2.5, 25.0, 50.0ug.ml-' were7.4, 10.7, 34-2, 36.6, 38.5, 42.3 percent respectively. (5).TS mANA werereduced about 44%, 47%, 46%, 62%and 73% after the cancer cells exposed- 6 -to 0.6, 1.2, 5.0, 10.0 and 20.0 ug.mr?nolatrexed dihydrochloride , the amplification product were 485bp and 570bp.CONCLUSIONS (1 ).Antiproliferative effect of nolatrexed dihydrochlorid... |
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