摘要
中、晚幼粒细胞及一些杆状和分叶核细胞。流式细胞分析术表明，SLM9123(10~(-6)M)处理HL-60细胞24小时可阻断细胞由G_1期向S期移行，作用5天可使G_1期细胞比例达70％。分子生物学实验结果显示，SLM9123(10~(-6)M)作用24小时，可使癌基因c-myc表达明显下降，c-fos基因表达水平提高。说明SLM9123在转录水平上调控参与细胞增殖分化的癌基因，发挥其分化诱导作用。
研究还发现SLM9123对另一株具有典型t(15；17)易位特征的人急性早幼粒白血病NB4细胞也有明显的分化诱导作用。SLM9123(10~(-6)M)能抑制NB4细胞的生长，使细胞的NBT还原能力及酸性磷酸酶活性显著提高，10~(-6)M作用6天可使NBT阳性细胞数达95％以上。SLM9123对NB4细胞形态影响出现较早，10~(-6)M作用2天可见核缩小，核质比减少；但形态分化不完全，10~(-6)M处理6天大部分细胞仍处于中幼或晚幼粒阶段，无杆状及分叶核细胞。
SLM9123还能诱导HL-60细胞产生程序性细胞死亡(apoptosis)。SLM9123低浓度(10~(-10)～10~(-8)M)作用5天，可使细胞核染色体断裂、核固缩，并出现凋亡小体(apoptotic body)；提取细胞DNA进行琼脂糖凝胶电泳呈现典型的DNA梯(DNA ladder)。用流式细胞仪进行细胞动力学研究可见，在G_1峰前面出现一个DNA含量减少的“G_1亚峰”(sub-G_1 peak)。Dot blot结果表明，SLM9123(10~(-6)M)处理HL-60细胞24小时后，使细胞内bcl-2基因表达显著降低，p53基因表达水平升高。说明SLM9123诱导细胞程序性死亡的作用机制与调节apoptosis相关基因的表达水平相关。
本论文建立了测定活细胞~(31)P NMR谱的方法，并用于癌细胞的分化诱导研究。结果表明，SLM9123和RA在诱导HL-60细胞和NB4细胞向粒细胞方向分化的同时，细胞内磷酸单酯(PME)、磷酸双酯(PDE)及ATP水平均有显著升高，细胞内PH值有所降低。说明SLM9123在诱导HL-60和NB4细胞分化的过程中，使细胞的磷脂代谢和能量代谢水平明显提高。
用高效液相色谱(HPLC)测定了SLM9123在大鼠血清中药代动力学特点。证明大鼠静脉注射10mg／kg SLM9123后，血清中药物浓度经一次加权的二室模型拟合，T1／2α为0.381h，T1／2β为12.625h，Vc为0.318L／kg，AUC为141.764μg·h／ml，CLs为0.071L／kg·h。研究发现SLM9123在大鼠体内有代谢产物，并作了初步鉴定。分别获得SLM9123 po和ⅳ给药后代谢物的药时曲线。为了研究SLM9123是否通过胎盘屏障进入胎鼠，对妊娠第11天的小鼠一次口服给约200mg／kg的SLM9123，给药后2-10h分别用HPLC检测母鼠血清及胎鼠匀浆的乙酸乙酯提取物，两者中均未发现原型药，在母鼠血清中检测到代谢物，而胎鼠中没有检测到。推测孕鼠口服SLM9123后未通过胎盘，可能与其无明显致畸作用有关。
SLM9123不论口服或腹腔给药对维甲类敏感的大鼠软骨肉瘤都具有明显的抑制作用，口服或腹腔给药18次(50mg／kg)抑制率分别为43.4％和62.1％。用MTT方法观察到SLM9123对培养的人卵巢癌A2780，人食管癌CaEs-17，人口腔癌KB及其耐药株KB／VCR200只在10~(-4)M高浓度时具有一定的生长抑制作用，但细胞毒作用程度较低。
SLM9123的初步毒性实验结果表明，腹腔给药对小鼠的LD50为4g／kg，口服给药对小鼠的LD50大于5g／kg，未见明显毒性反应。说明SLM9123毒性较低。
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 G_1 phase and that the cell population in S and G_2/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 ~(31)p 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 ~(31)p 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 EC_(50) 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 (T_(1/2α) and T_(1/2β)) 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 LD_(50) 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.
引文
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