Molecular and pharmacological studies of magnolol and honokiol on 1,25-Dihydroxyvitamin D3-induced human leukemia cell differentiation
厚朴酚與和厚朴酚對活化型維生素 D3 誘發之白血病細胞分化的分子藥理學研究
Student thesis: Doctoral Thesis
Leukemia cells are the results of blockage of the haematopoietic differentiation program that can be reactivated by several agents. Differentiation of several human myeloid cell lines could be induced by 1,25-dihydroxyvitamin D3 (VD3), however, the clinical use of VD3 is hampered by its hypercalcemic effect. One approach to overcome these problems is to introduce a second chemical that enhances the differentiation-inducing effects of VD3 and allows the use of the inducers at lower, non-toxic doses. It has been suggested that anti-inflammatory agents can enhance the terminal differentiation of induced leukemia cells. Magnolol (MG) and honokiol (HK), two lignans from Chinese medical herbs Magnolia officinalis (Houpu), have been observed to exert a variety of anti-inflammatory properties in different experimental models. Based on these findings, the effects of MG and HK on the differentiation of HL-60 human promyelocytic cells and in particular their potential effects on enhancing the activities of low doses of VD3 were investigated. MG and HK at µM levels were found to enhance differentiation of HL-60 cells induced by low dose of VD3 and another differentiation-inducing agent all-trans-retinoic acid (ATRA), as evidenced by nitroblue reduction assay, cell surface markers analysis, G0/G1 cell cycle blockage and expression of cyclin inhibitor p27. Mechanistically, evidence was provided to show the MEK/MAP kinase signaling pathway is involved in the actions of MG and HK. Interestingly, a more pronounced enhancement effect on differentiation was observed when MG and HK were simultaneously added to VD3 or ATRA. Moreover, MG and HK enhanced the cytotoxic effects of VD3 in VDR-positive cells MCF-7 but not in VDR-negative MDA-MB-231 cells, indicating that these effects are VDR dependent. Collectively, it is evident that MG and HK are potential differentiation enhancing agents which may allow the use of low doses of VD3 and ATRA in the treatment for acute promyelocytic leukemia. Nuclear factor kappa B (NF-B) is a transcription factor which regulates the transcription of a wide array of gene products that are involved in the cancer development, inflammation, immune response, apoptosis and cell proliferation. The classic form of NF-B is the heterodimer of the p50 and p65 subunits, which contains the transcriptional activation domain and sequestered in the cytoplasm as an inactive complex by the inhibitory proteins IBs. Recently, there is evidence that NF-B represses the transcriptional activities of vitamin D receptor (VDR). Marked differentiation enhancing effects were observed in HL-60 cells under the combined treatment of VD3 and NF-B inhibitors. Therefore, the effects of MG and HK on NF-B activities were firstly demonstrated in tumor necrosis factor alpha (TNF-) -activated cells. TNF--induced NF-B activation was blocked by MG and HK in different cancer cell lines including HL-60. MG and HK did not directly affect the NF-B-DNA binding. Instead, MG and HK suppressed the intrinsic and TNF--stimulated upstream IB kinases (IKKs) activities, suggesting a critical role of MG and HK in abrogating the phosphorylation and degradation of IB. Further studies on NF-B responses during MG/HK-induced differentiation enhancement in VD3-treated HL-60 cells were performed. Surprisingly, transient NF-B activation was observed in HL-60 cells treated with VD3 alone and in combination with MG/HK. To explore the NF-B responses during VD3-induced differentiation, systematic time slot studies were performed. Interestingly, a biphasic response of NF-B in VD3-treated leukemic HL-60 cells was observed. The biphasic NF-B response coincides with early down-regulation of NF-B and delayed re-activation of NF-B with the up-regulation of NF-B regulated gene. The subsequent stimulation of NF-B was observed concurrently with various stages of subunit p65 activation including residues S276 and S536 phosphorylation, nuclear translocation, and recruitment to NF-B gene promoters. It is suggested that the early suppression of NF-B activity could be the results of the IB induction, which requires de novo protein synthesis and is independent on IKK, Akt and MAPK pathways. VD3 also induced phosphorylation of IB via Akt-dependent IKK pathway, which leds to re-activation of NF-B signal and transient NF-B-regulated gene expressions. Moreover, an early elimination of p65 from vitamin D responsive element (VDRE) of CD14 and CAMP promoters was observed, suggesting that the transient NF-B inactivation may facilitate the initial VDR’s function. Notably, MG and HK were observed to enhance the IB induction, implying the possibilities that the differentiation enhancement could be caused by early NF-B inactivation. This study provides a new insight to the NF-B responses in VD3-treated cells and the exploitation of such findings may lead to the development of new VD3 analogs and VD3 combination agents for human inflammatory diseases and caner.
- Cell differentiation, Anti-inflammatory agents, Leukemia, Vitamin D