【作者】 何小庆；

【导师】 韩锐；

【作者基本信息】 中国协和医科大学 ， 肿瘤药理学， 1994， 博士

【摘要】 3’、5’-二叔丁基-4’-甲氧基-4-羧基查尔酮(R₉₁₅₈)是我所研制的第三代维甲类化合物。本文就R₉₁₅₈对癌细胞的分化诱导作用、分子药理学作用特点、作用机制及毒性进行了系统的研究。研究结果表明：R₉₁₅₈对人急性早幼粒白血病HL-60细胞具有明显的分化诱导作用。R₉₁₅₈ 10^-6mol／L可明显抑制HL-60细胞的生长及克隆形成率，并使成熟分化指标：NBT还原能力明显升高，10^-6mol／L作用6天可使NBT阳性细胞数达90％以上。细胞形态向成熟方向分化，镜下可见大量中晚幼粒细胞及一些杆状及分叶核细胞。电镜下细胞膜系统完整、核浆比例变小、胞浆中有特异性颗粒出现。流式细胞分析表明R₉₁₅₈ 10^-7mol／L处理HL-60细胞48小时就可阻断细胞由G1向S期移行。96小时可使G1期细胞数达78.2％。研究还发现R₉₁₅₈同样可使另一株人急性早幼粒白血病NB₄细胞生长抑制，NBT还原能力增强。R₉₁₅₈与RA诱导NB₄细胞分化的ED₅₀分别为3.9×10^-9及2.8×10^-9mol／L。R₉₁₅₈对NB₄细胞形态的影响出现较早，诱导2天就可见核缩小，核浆比例变小，但形态分化不完全，10^-6mol／L处理6天，大部分细胞仍集中在中幼或晚幼粒阶段，无杆状及分叶核细胞。NB₄细胞染色体呈亚四倍体型，染色体数分布在71-99之间，G显带表明它具有明显急性早幼粒白血病（APL）病人的t（15；17）移位特征。给R₉₁₅₈及RA 10^-6mol／L处理细胞3-6天后可使染色体非整倍体数目有所下降。从对照细胞的80.85±7.11降至RA：68.5±7.01（P＜0.01），R₉₁₅₈ 72.96±6.9（P＜0.01）。但t（15；17）移位未见消失。R₉₁₅₈对体外培养的人肝癌Bel-7402细胞也有良好分化诱导作用。R₉₁₅₈处理后可使生长抑制，克隆形成率下降。其作用强于RA。R₉₁₅₈及RA10^-5mol／L处理Bel₇₄₀₂细胞不同时间，可见肝癌的增殖指标甲胎蛋白（AFP）分泌及γ-谷胺酰转肽酶（γ-GT）活性下降，而肝癌分化指标白蛋白（Alb）分泌明显升高。R₉₁₅₈10^-6及10^-5mol／L处理48小时，形态上就可见核浆比例变小，继续处理可使核明显缩小，核固缩甚至碎裂，胞浆空泡化。但膜系统仍完整。利用[³H]TdR、[³H]Leu前体参入实验发现R₉₁₅₈对Bel-7402细胞DNA及蛋白质的合成均有一定抑制作用，但对蛋白质的抑制作用出现早且作用强，RA与之相比对Bel-7402细胞前体参入的影响较小。R₉₁₅₈对大鼠软骨肉瘤具有非常强的抑制作用，1.2mg／kg给药4次抑制率达99.3％。R₉₁₅₈对维甲类不敏感的动物移植性肿瘤也有一定抗肿瘤作用。其中R₉₁₅₈5mg／kg给药3次可使B₁₆黑色素瘤抑制率达58.9％（P＜0.01）；6mg／kg给药5次对Lewis肺癌的抑制率达65％（P＜0.01）。肉瘤S₁₈₀，R₉₁₅₈11.8mg／kg给药3次也可使抑制率达36.5％（P＜0.01）。在体外用MTT方法观察了R₉₁₅₈对培养的人卵巢癌A₂₇₈₀，大食管癌CaEs-17，人肝癌Bel-7402，人巨细胞肺癌PLA-801C及PLA-801D，人口腔癌KB及人结肠癌HCT-8等细胞生长的影响。发现R₉₁₅₈对上述细胞作用的ED₅₀均在2-6.45×10^-6mol／L范围之间，说明R₉₁₅₈有较强的细胞毒作用，而RA对上述细胞无明显细胞毒作用。以上说明R₉₁₅₈具有较强的抗肿瘤作用。为了研究R₉₁₅₈作用的选择性，我们用MTT法比较了R₉₁₅₈对体外培养的正常细胞的作用。发现R₉₁₅₈对小鼠成纤维Balb／3T3，中国仓鼠肺细胞CHL及人胚肺2BS细胞无明显细胞毒作用。R₉₁₅₈在明显抑制HL-60细胞克隆形成率的剂量下对小鼠骨髓细胞CFU-GM形成影响很小。用淋巴母细胞体外转化实验发现R₉₁₅₈对淋转无抑制作用。这说明R₉₁₅₈对正常组织毒性小，对免疫及造血组织无明显抑制作用，其抗肿瘤作用具有一定选择性。耐药与多药耐药是肿瘤化疗的一大难点。研究发现，R₉₁₅₈对多药耐药细胞KB／VCR 200及HCT-8／VCR 2000与其亲本细胞KB，HCT-8具有同样敏感性。ED₅₀分别为4.36×10^-6mol／L与1.05×10^-6mol／L，说明R₉₁₅₈与常规化疗药无交叉耐药。R₉₁₅₈10^-5mol／L处理24小时可使上述耐药细胞中高表达的mdrl多药耐药基因表达下降。但R₉₁₅₈对维甲酸耐药的HL-60／RA及维甲酸不敏感的K₅₆₂细胞与维甲酸具有交叉抗性。初步的毒性观察结果表明：R₉₁₅₈对小鼠的LD₅₀为118.3mg／kg，大鼠LD₅₀为19.36mg／kg。R₉₁₅₈在治疗剂量下对荷软骨肉瘤的大鼠，除使体重增加较慢外，血常规，肝、肾功能及各脏病理检查均未见异常。狗的初步亚急性实验发现R₉₁₅₈3mg／kg，口服15-21天可引起维A类过多症（如脱毛、体重下降等），但对血常规，心电图，肝肾功，在给药前中后无明显变化。病理报告除有肝脏充血，一条雄性狗睾丸曲细精管生精不良外，其它脏器均未见异常。运用基因重组技术研究发现，给维甲类不敏感的人髓样红白血病K₅₆₂细胞转入维甲酸受体基因hRAR_α，可大大增加其对RA及R₉₁₅₈的敏感性。用RA或R₉₁₅₈处理可使K₅₆₂-RAR_α细胞生长明显抑制，并使血红蛋白（Hb）的生成量增加。这提示R₉₁₅₈的作用机理与RA相似，通过维甲酸受体诱导肿瘤细胞分化。进一步用Dot blot技术研究发现R₉₁₅₈可在转录水平上调控某些基因的表达。R₉₁₅₈10^-6mol／L作用24小时，先于形态及生长速率变化就可使癌基因c-myc表达明显下降，而c-fos表达增高。在Bel-7402细胞，R₉₁₅₈10^-5mol／L处理24小时可见c-myc及N-ras癌基因表达下降，而诱导c-fos表达升高。这说明R₉₁₅₈的分子机制与RA相似，直接通过细胞核维甲酸受体，在转录水平上调控参与细胞增殖分化的癌基因，如c-myc，N-ras，c-fos等来完成其癌分化诱导作用。更多还原

【Abstract】 3’, 5"—ditertiary—butyl—4’—methoxy—4’—carboxyl chalcone （R9158）, a new retinoid, was developed by our institute. Through a routine screening program with human promyelocytic leukemia HL-60 cell line, it was found that R9158 has very strong inducing activity on differentiation of cancer cells. Systematic studies on the molecular pharmacology of R9158 were undertaken in this thesis.Results showed that R9158 significantly inhibits the growth and colony formation of promyelocytic leukemia HL-60 cells, while NBT-reduction （a functional differentiation marker of granulocytes） was dramatically augmented when the cells were exposed to R9158 at 10^-9-10^-6 mol/L, and the NBT positive cells reached about 90% at 10^-6 mol/L after 6 day exposure. Morphologically, HL-60 cells induced by R9158 matured along the granulocytic lineage. Flow cytometry demonstrated that R9158 as well as RA resulted in the arrest of cells in Gl phase and that the cell population in S and G2/M phase decreased significantly.NB4 cells, another promyelocytic leukemia cell line with a specific chromosome translocation t（15,17） was also studied. When the cells were exposed to R9158 or RA at a concentration of 10^-6 mol/L, cell proliferation was inhibited and NBT positive cells increased to 94% after 6 day exposure. The ED₅₀ of NBT-reduction was achieved at concentrations of 3.9×10^-9 mol/L and 2.8×10^-9 mol/L for R9158 and RA, respectively. Morphologically, most of the cells induced by R9158 or RA at 10^-6 mol/L were in the myelocytic or metamyelocytic stage and few cells were in the banded or segmented stage. Cytogenetic analysis revealed that the karyotype of NB4 cells was subtetraploid, and the chromosome numbers were in the range of 71-99. When NB4 cells were treated by R9158 or RA for 3-6 days, the abnormal poryploid chromosome numbers decreased to 72.9±6.9 （P＜0.01） or 68.5±7.01 （P＜0.01）, but the t（15,17） translocation did not disappear.Cell proliferation and colony forming efficiency of the human hepatocarcinoma, Bel-7402 cell line were significantly inhibited when R9158 or RA were added at concentrations of 10^-6-10^-5 mol/L for 2-6 days. However, R9158 was more efficient. Secretion of alpha-fetoprotein （AFP） and the activity ofγ-glutamyl transpeptidase （γ-GT）, the hepatoma proliferation marker, were decreased, while the secretion of albumin （ALB）, the hepatoma differentiation marker, was enhanced dramatically. These results suggest that R9158 induced Bel-7402 cell differentiation. It was also found that the ratio of nucleus to cytoplasm decreased when cells were exposed to R9158 (10^-6-10^-5 mol/L) for 2-4 days. If the cells were treated continuously for 8 days, the nucleus became pyknotic or fragmented and the cytoplasm became vacuolated. [³H]-TdR and [³H]-Leu incorporation into DNA and protein was also inhibited by R9158. The inhibition of protein synthesis appeared earlier and was more effective than DNA synthesis inhibition.When R9158 was given orally to rats bearing chondrosarcoma, at the dosage of 1.2mg/kg or 0.6 mg/kg for 4 times over the entire course of the experiment, this compound significantly inhibited the growth of the tumor and the inhibition rate reached 99.3％or 95.6％, respectively. It is interesting to note that R9158 also inhibited the growth of other transplantable tumors, such as Lewis lung carcinoma, sarcoma 180, and melanoma B16, in mice. In vitro studies demonstrated that R9158 elicited moderate cytotoxic effects on various human cancer cell fines. The EC₅₀ for ovarian cancer A2780, hepatoma Bel-7402, esophageal carcinoma CaES-17, lung giant cell carcinoma PLA-801C and PLA-801D, oral carcinoma KB, and colon carcinoma HCT-8 were all in the range of 2-6（?）10^-6 mol/L, while in some normal human and mice cell lines, the EC₅₀ for human embryo lung 2BS cell line is 1.70×10^-5 mol/L, for Chinese hamster lung CHL cell line is 3.24×10^-5 mol/L, and for mouse fibroblast balB/3T3 cell fine is 1.98×10^-5 mol/L. R9158 can also dramatically inhibit the colony formation efficiency of HL-60 cells. In contrast, R9158 has little effect on the CFU-GM of bone marrow cells in mice at the same concentration. The results demonstrate that R9158 has a higher selectivity for cancer cells. Cross resistance studies showed that RA resistant cell line （HL-60/RA） is cross resistant to R9158; however, in multidrug resistant cell lines of KB/_VCR200 or HCT-8/_VCR2000, the results showed that they had the same sensitivity as parent KB or HCT-8 cells. Dot blot analysis showed that the expression of multidrug resistant gene, mdrl, was decreased when cells were exposed to R9158 orRA at 10^-6 mol/L for 24 hours.Toxicity studies demonstrated that the LD₅₀ of R9158 for mice is 118.3 mg/kg and 19.36 mg/kg for rats. The preliminary subacute toxicity studies on dogs suggested that 3 mg/kg of R9158 once daily for 15-21 days caused side effects which were similar to hypervitaminosis A, such as hair and body weight loss. However, no myelosuppression, hepatic damage, or nephrotoxicity was found.Retinoids are thought to act through nuclear retinoic acid receptors which in turn act on some target genes. Dot blot analysis of total RNA with cDNA probes specific to hRARα, hRARβ, and hRARγindicated that HL-60 or HL-60/RA cells all have a basal expression level of RARα, which change little even when treated with RA or R9158. However, RARβand RARγexpression was enhanced in Bel-7402 cells when they were treated by R9158 or RA at 10^-5 mol/L for 24 hours. Further studies revealed that RA resistant K562 cells showed cross resistance to R9158. but when RARαcDNA was transfected into K562 cells by a retroviral vector, the two drugs could significantly decrease the cell proliferation. Additional dot blot analysis indicated that R9158 inhibited c-myc expression in HL-60 cells and inhibited both c-myc and N-ras expression in Bel-7402 cells. The expression of c-los in both of these cell lines were enhanced by R9158. In summary, the results clearly, demonstrated that R9158 modulates oncogene expression and induces cell differentiation through retinoic acid receptors.更多还原