PTC-209 hydrobromide (PTC-209 HBr) is the hydrobromide salt of PTC-209, which is a potent and selective BMI-1 inhibitor with IC50 of 0.5 μM, and results in irreversible reduction of cancer-initiating cells (CICs).

BMI1:0.5 μM

PTC-209在人类结直肠癌细胞HCT116和人类纤维肉瘤细胞HT1080中同时抑制UTR介导的报告基因表达和内源性BMI-1表达。PTC-209通过BMI-1依赖的方式减缓结直肠肿瘤细胞生长。此外，PTC-209还通过不可逆的生长抑制作用损害结直肠癌起始细胞(CICs)。[1]

PTC-209（60 mg/kg/天，皮下注射）有效抑制了肿瘤组织中BMI-1的产生，并在带有初级人类结肠癌异种移植物、人类结肠癌细胞系LIM1215或HCT116异种移植物的小鼠中停止了已建立肿瘤的生长。PTC-209还在体内减少了功能性结直肠CICs的频率。[1]

Untranslated region-mediated luciferase reporter expression: HEK293 cells are transfected with a GEMS reporter vector that contains the luciferase open-reading frame flanked by and under post-transcriptional control of the BMI-1 5′ and 3′ UTRs. The resulting stable cells (F8) are treated with PTC-209 or vehicle control overnight, and then luciferase reporter activity is determined using Bright-Glo assays. The assays are run in triplicate for each point, and the percentage of inhibition was calculated against vehicle control.

To determine whether pretreatment with the inhibitor affects tumor cell growth, cells are plated with the inhibitor for 4 d in vitro and plated in limiting doses in vitro without adding further inhibitor. Trypan blue exclusion is used to count viable cells. The in vitro sphere-initiating cell frequency is calculated after inhibitor treatment by evaluating the number of wells containing spheres. For the experiments where LDAs are set up following recovery of PTC-209 treated cells, 6-well plates were seeded with 1E6 cells per well and incubated overnight. Cells are subsequently treated for 4 d in triplicate with either DMSO vehicle or PTC-209 (0.01, 0.1, 1 and 10 μM). Drug treatments are washed off and 4 mL fresh suspension medium added to all wells. To assess cell viability following the 4 d treatment window, cells are trypsinized and counted at 0, 24, 72 and 120 h after removal of the drug. Long-lasting effects of the drug treatment on sphere-forming ability are assessed by plating LDAs (50,000, 10,000, 1,000,100, 10 and 1 cell per well) using the cells obtained 120 h after the 4-d drug treatment.(Only for Reference)