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

1-（3，5-二氯-2，6-二羟基-4-甲氧基苯基）-1-己酮（DIF-1）是盘状网壳菌（Dictyosterium discoideum）中的一种诱导柄细胞生长的因子。最近，发现DIF-1在大鼠胰腺肿瘤AR 42 J细胞中表现出抗肿瘤活性并抑制细胞生长，并且在人白血病K562细胞中诱导红系分化。此外，已知该分子在治疗的早期阶段增加细胞中的胞质钙浓度。在本项目中，我们首先关注非钙调神经磷酸酶（CaN），因为该蛋白在细胞的钙信号传导中起重要作用。DIF-1和DIF-2在较低浓度（10-30 nM）下激活CaN。但在较高浓度（10-30 μM）时，化合物对酶的抑制作用是立体专一性的。这些结果表明CaN是DIF的靶蛋白。接下来，我们检查了DIF-1无Akt/PKB在K562细胞中的作用。在与5-30 μM DIF-1孵育数小时内，激酶被磷酸化并被有效激活。降钙剂TMB-8和EGTA与A23187一起抑制DIF-1诱导的Akt/PKB活化。DIF-1诱导胰岛素分泌型胰岛素瘤细胞（INS-1）凋亡的剂量依赖性。我们发现细胞凋亡是不依赖Caspase的。我们研究了DIF-1对一些细菌和真菌以及流感病毒的影响。DIF-1对细菌和真菌无明显抑制作用，但对MDCK细胞中病毒空斑的形成有剂量依赖性抑制作用，IC_（50）为18.7 μM。

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.