MDK83190 (Apoptosis Activator 2) is a potent apoptosis activator, induceing caspase-3 activation, PARP cleavage, and DNA fragmentation .

Leukemia origin cells:4-9 uM (IC50)

Apoptosis Activator 2 (20 μM) 在降低的cyto c浓度下，将Apaf-1在细胞凋亡体中的比例增加1.5倍至33%。同时，该化合物在降低的cyto c水平和caspase-3激活水平下，使caspase-3活化程度增加4倍。Apoptosis Activator 2强烈诱导caspase-3活化、PARP裂解和DNA片段化，最终以4 μM的IC50杀死细胞。Apoptosis Activator 2诱导PBL、HUVEC、Jurkat、Molt-4、CCRF-CEM、BT-549、MDA-MB-468和NCI-H23细胞凋亡，其IC50分别为50 μM、43 μM、4 μM、6 μM、9 μM、20 μM、44 μM和35 μM。在48个测试的肿瘤细胞系中，Apoptosis Activator 2对绝大多数肿瘤细胞系展现出细胞静态效应，以10 μM的浓度在40个细胞系中抑制细胞生长50-100%。[1] Apoptosis Activator 2通过触发细胞凋亡体的形成引起细胞死亡。En1表达水平对Apoptosis Activator 2处理的腹侧中脑培养存活率无显著影响（-8.1 ± 6.0%）。如果使用其他三种试剂，存活率也没有显著变化（-10.7 ± 4.7%）。[2] Apoptosis Activator 2（10 μM）通过诱导凋亡DNA梯度和Tunel实验评估，在AGS细胞中诱导凋亡，并增强了抗TROP2偶联脂质体的凋亡诱导作用。[3] Cyclohexamide（10 μg/mL）或zVAD（50 μM）显著保护神经细胞培养物免受Apoptosis Activator 2的毒性。Apoptosis Activator 2（3 μM）导致大量神经元出现细胞死亡涉及的凝集核。DHT（10 nM）或E2（10 nM）显著保护神经细胞培养物免受Apoptosis Activator 2的毒性。[4]

Cell-Free Apoptosis Assay: HeLa cell cytoplasmic extracts are prepared according to previously published reports. Apoptosis Activator 2 in DMSO are distributed into 96-well microtiter plates at a final concentration of 1 mM (final DMSO concentration is 1% vol/vol). To each well is added 250 μg of total protein from cytoplasmic extracts in HEB buffer (50 mM Hepes, pH 7.4/50 mM KCl/5 mM EGTA/2 mM MgCl), with 2 mM DTT, 2 μM cyto c, and 0.5 μM DEVD-AFC (Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin) substrate in a total of 150 μL. Plates are incubated at 37 ℃, and fluorescence is read in a LJL Biosystems plate reader at 10-min intervals.

All viable cells within the defined field of a microscope reticle grid are counted using a manual mechanical counter by an experimenter blinded to condition. Cells are scored viable on the basis of both positive staining with the vital dye calcein acetoxymethyl ester and the morphological criterion of a smooth, spherical soma. Counts of viable cells are made in four non-overlapping fields per culture well with each condition represented by 3 separate wells. The number of viable cells counts per well for vehicle-treated control conditions ranged from 100-200. All experiments are repeated in at least 3 independent culture preparations. Raw cell count data are statistically analyzed with one-way ANOVA, followed by between group comparisons using the Fisher LSD test (significance indicated by P < 0.05). Cell viability is presented graphically as a percentage of live cells in the vehicle-treated control condition.(Only for Reference)