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Crocetin, a carotenoid isolated from the seeds of Gardenia jasminoides, was found to be a potent inhibitor of tumor promotion induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse skin. When mouse fibroblast NIH/3T3 cells were treated with TPA alone, protein kinase C (PKC) translocated from the cytosolic fraction to the particulate fraction. Pretreatment with 60 and 120 microM crocetin for 15 min inhibited the TPA-induced PKC activity in the particulate fraction by 50% and 66%, respectively, but did not affect the level of PKC protein. Crocetin also reduced the level of TPA-stimulated phosphorylation of cellular proteins. Cells pretreated with crocetin (120 microM) had 55% less PKC [3H]phorbol dibutyrate-binding capacity. Suppression of TPA (100 ng/mL)-induced c-jun and c-fos gene expression was also observed in the mouse fibroblast cells pretreated with crocetin (30, 60, and 120 microM). Our results provided a basis for understanding the inhibitory effect of crocetin on TPA-mediated tumor promotion.

SUMMARY:In this study, using zymogram analysis two proteolytic activities were identified in the mouse sarcoma 180 (S-180) cells that were activated by trypsin treatment and inhibited by both BBI and ACTI. These enzymes, with molecular weight s of 46 kDa (dominant band) and 62 kDa (minor band), were mainly localized in the cytosol, and had optimal activity at pH 7 and 8 respectively. Their inhibition by DFP, BBI and ACTI but not EDTA and TPCK indicated they were trypsin-like serine proteases and may be the intracellular target-enzymes of protease inhibitors. The level of the precursor of the 62 kDa protease was significantly increased in the S-180 solid and soft tumors, whereas the level of the 46 kDa precursor was almost undetectable, implying that a physiological role may be played by these serine proteases during tumor invasion.