Siu-Caldera M L, Clark J W, Santos-Moore A, Peleg S, Liu Y Y, Uskoković M R, Sharma S, Reddy G S
Dept. of Pediatrics, Women and Infants' Hospital of Rhode Island, Brown University School of Medicine, Providence 02905, USA.
J Steroid Biochem Mol Biol. 1996 Dec;59(5-6):405-12. doi: 10.1016/s0960-0760(96)00134-3.
1alpha,25(OH)2-16-ene-D3, a synthetic analog of the steroid hormone, 1alpha,25(OH)2D3, has great potential to become a drug in the treatment of leukemia and other proliferative disorders, because of its minimal in vivo calcemic activity associated with a potent inhibitory effect on cell growth. However, at present, the mechanisms through which 1alpha,25(OH)2-16-ene-D3 expresses its biological activities are still not completely understood. Our previous in vitro study in a perfused rat kidney indicated for the first time that 1alpha,25(OH)2-16-ene-D3 and 1alpha,25(OH)2D3 are metabolized differently. 1alpha,25(OH)2-24-oxo-16-ene-D3, an intermediary metabolite of 1alpha,25(OH)2-16-ene-D3 formed through the C-24 oxidation pathway, accumulated significantly in the perfusate when compared to 1alpha,25(OH)2-24-oxo-D3, the corresponding intermediary metabolite of 1alpha,25(OH)2D3. In a subsequent in vivo study, we also reported that 1alpha,25(OH)2-24-oxo-16-ene-D3 exerted immunosuppressive activity equal to its parent, without causing significant hypercalcemia. In order to establish further the critical role of 1alpha,25(OH)2-24-oxo-16-ene-D3, in generating some of the key biological activities ascribed to its parent, we performed the present in vitro study using a human myeloid leukemic cell line (RWLeu-4) as a model. Comparative target tissue metabolism studies indicated that 1alpha,25(OH)2-16-ene-D3 and 1alpha,25(OH)2D3 are metabolized differently in RWLeu-4 cells, and the differences were similar to the ones we previously observed in the rat kidney. The significant finding was the accumulation of 1alpha,25(OH)2-24-oxo-16-ene-D3 in RWLeu-4 cells because of its resistance to further metabolism. Biological activity studies indicated that both 1alpha,25(OH)2-16-ene-D3 and its 24-oxo metabolite produced growth inhibition and promoted differentiation of RWLeu-4 cells to the same extent, and these activities were several fold higher than those exerted by 1alpha,25(OH)2D3. In addition, the genomic action of each vitamin D compound was assessed in a rat osteosarcoma cell line (ROS 17/2.8) by measuring its ability to transactivate a gene construct containing the vitamin D response element of the osteocalcin gene linked to the growth hormone reporter gene. In these studies, both 1alpha,25(OH)2-16-ene-D3 and its 24-oxo metabolite exerted similar but potent transactivation activity which was several fold greater than that exerted by 1alpha,25(OH)2D3 itself. In summary, our results indicate that the production and slow clearance of the bioactive intermediary metabolite, 1alpha,25(OH)2-24-oxo-16-ene-D3, in RWLeu-4 cells contributes significantly to the final expression of the enhanced biological activities ascribed to its parent analog, 1alpha,25(OH)2-16-ene-D3.
1α,25(OH)₂ - 16 - 烯 - D₃是类固醇激素1α,25(OH)₂D₃的一种合成类似物，由于其体内降钙活性最小且对细胞生长具有强大的抑制作用，因此在治疗白血病和其他增殖性疾病方面具有很大的成为药物的潜力。然而，目前1α,25(OH)₂ - 16 - 烯 - D₃发挥其生物活性的机制仍未完全明确。我们之前在灌注大鼠肾脏中的体外研究首次表明，1α,25(OH)₂ - 16 - 烯 - D₃和1α,25(OH)₂D₃的代谢方式不同。1α,25(OH)₂ - 24 - 氧代 - 16 - 烯 - D₃是1α,25(OH)₂ - 16 - 烯 - D₃通过C - 24氧化途径形成的中间代谢产物，与1α,25(OH)₂D₃的相应中间代谢产物1α,25(OH)₂ - 24 - 氧代 - D₃相比，它在灌注液中显著积累。在随后的体内研究中，我们还报告称，1α,25(OH)₂ - 24 - 氧代 - 16 - 烯 - D₃发挥的免疫抑制活性与其母体相当，且不会引起明显的高钙血症。为了进一步确定1α,25(OH)₂ - 24 - 氧代 - 16 - 烯 - D₃在产生归因于其母体的一些关键生物活性方面的关键作用，我们以人髓系白血病细胞系（RWLeu - 4）为模型进行了本体外研究。比较性靶组织代谢研究表明，1α,25(OH)₂ - 16 - 烯 - D₃和1α,25(OH)₂D₃在RWLeu - 4细胞中的代谢方式不同，这些差异与我们之前在大鼠肾脏中观察到的相似。重要的发现是1α,25(OH)₂ - 24 - 氧代 - 16 - 烯 - D₃在RWLeu - 4细胞中积累，因为它对进一步代谢具有抗性。生物活性研究表明，1α,25(OH)₂ - 16 - 烯 - D₃及其24 - 氧代代谢产物对RWLeu - 4细胞产生生长抑制并促进分化的程度相同，且这些活性比1α,25(OH)₂D₃所发挥的活性高几倍。此外，通过测量每种维生素D化合物激活含有与生长激素报告基因相连的骨钙素基因维生素D反应元件的基因构建体的能力，在大鼠骨肉瘤细胞系（ROS 17/2.8）中评估了它们的基因组作用。在这些研究中，1α,25(OH)₂ - 16 - 烯 - D₃及其24 - 氧代代谢产物均发挥了相似但强大的反式激活活性，该活性比1α,25(OH)₂D₃本身所发挥的活性高几倍。总之，我们的结果表明，生物活性中间代谢产物1α,25(OH)₂ - 24 - 氧代 - 16 - 烯 - D₃在RWLeu - 4细胞中的产生和缓慢清除对归因于其母体类似物1α,25(OH)₂ - 16 - 烯 - D₃的增强生物活性的最终表达有显著贡献。
