Markaverich B, Varma M, Densmore C, Tiller A, Schauweker T, Gregory R
Int J Oncol. 1994 Jun;4(6):1291-300. doi: 10.3892/ijo.4.6.1291.
Previous studies from this laboratory demonstrated that 2,6-bis-([3,4-dihydroxyphenyl]methylene)cyclohexanone (BDHPC) and related compounds mimic methyl p-hydroxyphenyllactate (MeHPLA) as endogenous ligands for nuclear type II [H-3]estradiol binding sites. Occupancy of type II sites by these agents results in the inhibition of malignant cell proliferation in vitro and mammary tumor growth in vivo. The present studies were designed to assess the effects of BDHPC esterification on type II site binding interactions in uterine nuclei and in cultured MCF-7 human breast cancer cells in vitro. The results of these experiments demonstrate that in rat uterine nuclear fractions BDHPC acetate (Kd approximately 100 nM) interacts with type II sites with a 100-fold lower affinity than BDHPC (Kd approximately 0.9 nM) and BDHPC benzoate failed to inhibit [H-3]estradiol binding under these experimental conditions. Conversely, BDHPC and BDHPC acetate displayed very similar binding affinities for type II sites in cultured MCF-7 human breast cancer cells and there was a direct correlation between nuclear type II site occupancy and the inhibition of cellular proliferation by these two compounds. BDHPC benzoate failed to interact with type II sites or inhibit MCF-7 cell proliferation. Taken together, these results suggested that BDHPC acetate, but not BDHPC benzoate, was being hydrolyzed by esterases in MCF-7 cells, releasing the free parent compound. This conclusion was supported by the observations that incubation of BDHPC acetate in mammary tumor cytosol preparations resulted in essentially quantitative hydrolysis to BDHPC as determined by thin layer chromatography (TLC) and by high performance liquid chromatography (HPLC) analysis of tumor cytosol extracts. Conversely, BDHPC benzoate was not hydrolyzed by tumor esterases which is consistent with the inability of this compound to bind to type II sites or inhibit MCF-7 human breast cancer cell proliferation. These experiments confirm and extend the hypothesis that esterase hydrolysis of MeHPLA related compounds represents an important biological step involved in the control of the biological activity of type II site agonists which appear to regulate malignant cell proliferation through this binding interaction.
本实验室之前的研究表明，2,6-双（[3,4-二羟基苯基]亚甲基）环己酮（BDHPC）及相关化合物可模拟对羟基苯乳酸甲酯（MeHPLA），作为核II型[H-3]雌二醇结合位点的内源性配体。这些试剂占据II型位点会导致体外恶性细胞增殖受到抑制，以及体内乳腺肿瘤生长受到抑制。本研究旨在评估BDHPC酯化对子宫细胞核以及体外培养的MCF-7人乳腺癌细胞中II型位点结合相互作用的影响。这些实验结果表明，在大鼠子宫核组分中，乙酸BDHPC（解离常数约为100 nM）与II型位点相互作用的亲和力比BDHPC（解离常数约为0.9 nM）低100倍，且在这些实验条件下，苯甲酸BDHPC未能抑制[H-3]雌二醇的结合。相反，BDHPC和乙酸BDHPC对体外培养的MCF-7人乳腺癌细胞中II型位点显示出非常相似的结合亲和力，并且这两种化合物对核II型位点的占据与细胞增殖抑制之间存在直接相关性。苯甲酸BDHPC未能与II型位点相互作用或抑制MCF-7细胞增殖。综上所述，这些结果表明，乙酸BDHPC而非苯甲酸BDHPC在MCF-7细胞中被酯酶水解，释放出游离的母体化合物。这一结论得到了以下观察结果的支持：通过薄层色谱（TLC）以及对肿瘤细胞溶质提取物的高效液相色谱（HPLC）分析确定，乙酸BDHPC在乳腺肿瘤细胞溶质制剂中孵育后基本上定量水解为BDHPC。相反，苯甲酸BDHPC未被肿瘤酯酶水解，这与该化合物无法结合II型位点或抑制MCF-7人乳腺癌细胞增殖一致。这些实验证实并扩展了以下假设：MeHPLA相关化合物的酯酶水解是控制II型位点激动剂生物活性的一个重要生物学步骤，这些激动剂似乎通过这种结合相互作用来调节恶性细胞增殖。
