Identification of receptor of anti-tumor factor DIF and development of new DIF-analogs

1-(3，5-dichloro-2，6-dihydroxy-4-methoxyphenyl)-1-hexanone(DIF-1)是盘状网柄菌中的一种茎细胞诱导因子。最近发现DIF-1在大鼠胰腺癌AR42J细胞中具有抗肿瘤活性，抑制细胞生长，在人白血病K562细胞中具有诱导红系分化的作用。此外，已知该分子在治疗的早期阶段会增加细胞内的胞浆钙浓度。在本项目中，我们首先关注了NO钙调神经磷酸酶(CaN)，因为该蛋白在细胞的钙信号转导中起着重要作用。DIF-1和DIF-2在较低浓度(10-30 nm)激活CaN。但在较高浓度(10-30μM)时，这些化合物对酶活性具有立体特异性抑制作用。这些结果表明，CaN是DIF的靶蛋白。接下来，我们检测了DIF-1、NO、Akt/PKB在K562细胞中的作用。在与5-30μM DIF-1孵育的几个小时内，该激酶被磷酸化并被有效激活。钙还原剂TMB-8和EGTA与A23187共同抑制DIF-1诱导的Akt/PKB活化。DIF-1呈剂量依赖性诱导胰岛素分泌细胞(INS-1)细胞凋亡。我们发现，细胞的凋亡是由Caspase非依赖性诱导的。我们研究了DIF-1对一些细菌和真菌以及一种流感病毒的影响。DIF-1对细菌和真菌均无明显抑制作用，但对宿主细胞中病毒空斑的形成有抑制作用，且呈剂量依赖关系，IC_(50)为18.7μM。

1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone (DIF-1) is a stalk-cell-inducing-factor in a slime mold Dictyosterium discoideum. Recently, it was found that DIF-1 exhibits anti-tumor activities and suppresses cell growth in rat pancreatic tumor AR42J cells and that induces erythroid differentiation in human leukemia K562 cells. Furthermore the molecule is known to increase in cytosolic calcium concentration in the cells in the early stage of the treatment. In the present project, we firstly focused no calcineurin (CaN), since this protein plays an important role in the calcium signaling of the cells. DIF-1 and DIF-2 activated CaN in the lower concentration (10-30 nM). But in the higher concentration (10-30 μM) the compounds inhibited the enzyme activity stereo-specifically. These results suggest that CaN is a target protein for DIF. Next we examined the effect DIF-1 no Akt/PKB in K562 cells. The kinase was phosphorylated and potently activated within several hours of incubation with 5-30 μM DIF-1. Calcium-reducing agents TMB-8 and EGTA together with A23187 inhibited the DIF-1 induced activation of Akt/PKB. DIF-1 induced apotosis in insulin secreting insulinoma cells (INS-1) dose-dependently. We found that the apotosis was induced Caspase-independently. We examined the effects of DIF-1 on some bacteria and fungi, and an influenza virus. DIF-1 did not show marked effect no bacteria fungi tested, but it inhibited viral plaque formation in its host MDCK cells in a dose-dependent manner and IC50 was 18.7 μM.