ANTIPROLIFERATIVE ACTIVITY IN-VITRO AND IN-VIVO OF THE SPICAMYCIN ANALOG KRN5500 WITH ALTERED GLYCOPROTEIN EXPRESSION IN-VITRO

The spicamycin analogue KRN5500 (NSC 650426; SPA) is derived from Stre ptomyces alanosinicus. The unique structure contains a purine, an amin oheptose sugar, glycine, and a tetradecadiene fatty acid, SPA potently inhibits the growth of certain human tumor cell lines in vitro (IC50 for growth <100 nM) and displays marked activity in vivo in Cole 205 c olon carcinoma xenografts, Selective inhibition of labeled precursor i ncorporation was not evident at 1 or 4 h of exposure to the drug, but at 8 h. [H-3]leucine incorporation was inhibited by approximately 40% at or below the IC50 for cell growth, Because of the structural simila rity of SPA to inhibitors of glycoprotein processing, we examined the effect of SPA on indicators of glycoprotein synthesis and processing i n HL60TB promyelocytic leukemia and Cole 205 colon carcinoma cells, Br ief periods of exposure (similar to 30 min) to SPA at the IC50 for gro wth increased incorporation of [H-3]mannose. When examined by Western blotting after prolonged (40-48 h) incubation with lectins that target mannose-containing carbohydrates, Galanthus nivalis agglutinin and co ncanavalin A, a qualitative change in the pattern of mannose-containin g glycoproteins was observed in HL60TB cells. Significant changes in t he pattern of surface glycoprotein expression in intact cells were dem onstrated by flow cytometry using fluorescence-labeled lectins. An inc rease in the number of cells binding G. nivalis agglutinin (indicating terminal mannose) was noted, but a decrease in the amount of lectin b ound per cell was noted for wheat germ agglutinin (detecting sialic ac id and terminal beta-N-acetyl glucosamine residues), Electron microsco py revealed loss of microvilli, and the Golgi apparatus appeared infla ted, Our findings, therefore, raise the possibility that cells exposed to SPA have altered glycoprotein processing after exposure to low con centrations of drug, prior to the occurrence of overt cytotoxicity. Th ese effects are consistent with a prominent early effect of SPA on the enzymatic machinery or organelles important for proper glycoprotein p rocessing and emphasize the novelty of this agent's likely mechanism o f action.