| 3-Acetyl-5,7-diretinoyloxycoumarin (SLM9123), a new retinoid was developed by our institute. The cancer chemopreventive activity, the induction of differentiation and apoptosis of cancer cells and the mechanism of action of SLM9123 were studied in this thesis.The two-stage chemical carcinogenesis model was used in the chemopreventive activity studies. It was demonstrated that SLM9123 could inhibit DMBA/croton oil induced mouse skin papilloma, and prolong the latent period of tumor occurrence, decrease the incidence of papilloma and reduce tumor number per mouse in a dose-dependent manner. It was proved that SLM9123 is a potent cancer chemopreventive agent in vivo.SLM9123 exhibited a significantly inhibitory effect on cyclophosphamide (CTX)-induced micronucleus formation of polychromatic erythrocytes(PCE) of bone marrow in mice. In Ames assay, SLM9123 could decrease the number of TA97 His+-revertants induced by 4-nitroquinline N-oxide(4NQO) and TA98 induced by daunorubicin. The results suggested that the antimutagenic activity of SLM9123 may related to its protection of DNA damage and the repairment of DNA lesions in cells.Experiments showed that SLM9123 effectively inhibit croton oil-induced mouse ear edema and ornithine decarboxylase (ODC) activity of epidermis cells in mice. By using the scrape-loading and dye transfer (SLDT) technique, it was demonstrated that TPA inhibited the gap junction intercellular communication (GJIC), while SLM9123 could counteract the inhibition of TPA on GJIC and enhance GJIC in 2BS cells. The results revealed that SLM9123 has strong antipromotive activity.SLM9123 significantly inhibited the growth and colony formation of human promyelocytic leukemia HL-60 cells,while NBT-reduction and acid phosphatase activity (functional differentiation markers of granulocytes) were dramatically augmented when the cells exposed to SLM9123 at 10-8-10-6 mol/L, and the NBT positive cells reached about 90％at 10-6 mol/L after 6 day exposure. Morphologi- cally, HL-60 cells induced by SLM9123 differentiated along the granulocytic line- age. Flow cytometry demonstrated that SLM9123 exposure resulted in the arrest of cells in G1 phase and that the cell population in S and G2/M phase decreased sig- nificantly. Dot blot analysis indicated that SLM9123 inhibited c-myc expression and enhanced c-fos expression in HL-60 cells.NB4 cells, another human acute promyelocytic leukemia cell line with a spe- cific chromosome translocation t(15; 17) was also studied. When the cells were ex- posed to SLM9123 at a concentration of 10-6 mol/L, cell proliferation was in- hibited and NBT positive cells as well as acid phosphatase activity were increased significantly. After 6 day exposure at 10-6 mol/L, NBT positive cells reached to 95％. Morphologically, most of the cells induced by SLM9123 at 10-6 mol/L were in the myelocytic or metamyelocytic stage and few cells were in the banded or segmented stage.Apoptosis is an active process of programmed cell suicide and now is believed to play an important role in tumor chemotherapy. The apoptosis inducing effect of SLM9123 in HL-60 cells were studied. In agarose gel electrophoresis, DNA ex- tracted from HL-60 cells treated with 10-10-10-8 mol/L SLM9123 showed a typical internucleosomal DNA degradation, i. e. , DNA ladder and morphological changes as nuclear chromosome segmentation and condensation as well as apoptotic body. In addition, the characteristic apoptotic DNA peak of cells was revealed by flow cytometry. SLM9123 also inhibited bcl-2 and enhanced p53 expression in HL-60 cells treated with 10-6 mol/L for 24 hours.The method for 31p nuclear magnetic resonance (NMR) spectra of cells in sus- pension was established as a valuable means of noinvasive observation of high- energy phosphate-containing compounds and phospholipid metabolites in intact cell. The spectra indicated that the levels of phosphomonoester(PME), phosphodi- ester(PDE) and ATP were increasedin HL-60 and NB4 cells treated with SLM9123 or RA in a time-dependent manner, while the intracellular pH values decreased. The results demonstrate that 31p NMR reflects the biochemical changes associated with cell differentiation.When SLM9123 was given orally and peritoneally to rats bearing chondrosarcoma at the dosage of 50mg/kg for 18 times over the entire course of the experiment, this compound significantly inhibited the growth of the tumor and the inhibition rate reached 43.4％and 62.1％respectively. The EC50 for ovarian cancer A2780, esophageal carcinoma CaEs-17, oral carcinoma KB and VCR resistant cell line KB/VCR200 were all at the concentration of 10-4 mol/L. It is shown that SLM9123 elicited cytotoxic effects on these human cancer cell lines in vitro at very high concentration.The pharmacokinetics of SLM9123 in rats after single intravenous administra- tion was studied. Waters M244 HPLC system equipped with a M440 absorbance detector (wavelength 365nm) was used. After i. v. of SLM9123 10mg/kg, the dy- namic change of the concentration of SLM9123 in rat serum can be characterized as two phases, and the corresponding half-life times (T1/2α and T1/2β) were 0.381h and 12.625h respectively. The clearance of SLM9123 was 0.071 L/kg.h and AUC was 141.764 ug.h/ml. It was found that there was metabolite of SLM9123 in rat serum. Pregnant ICR mice were given a single oral dose (200mg/kg) on day 11 of gestation, and the metabolite of SLM9123 was detected in the maternal plasma during 2 to 10 hours after administration, while SLM9123 and its metabolite were not found in the embryo at the same time. Results suggested that SLM9123 does not go through the placenta, which may be related to the low teratogenic activity of SLM9123.Toxicity studies demonstrated that the LD50 of SLM9123 for mice is 4g/kg (i. p.) and no one died when given orally at 5g/kg. It showed that SLM9123 has very low toxicity.In summary, SLM9123 is a new attractive retinoid which has high activity but low toxicity and low teratogenicity.
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