Temsirolimus (CCI-779) is a specific mTOR inhibitor ( IC50: 1.76 μM), used in the treatment of advanced renal cell cancer.

mTOR:1.76 μM (cell free)

Temsirolimus (CCI-779; 10 nmol/L至<5 μmol/L) 主要展现了一种平坦且选择性的细胞增殖抑制作用。对Temsirolimus 敏感的细胞系，其增殖被抑制≥50%，包括LNCap、PC3MM2、MDA468、H446和Caco2；而抑制率≤25%的细胞系为SW480、HT29、HCT116和H460 [1]。经过3天100 nmol/L CCI-779处理后，PC-3和DU145细胞的集落形成数分别为0.18 ± 0.09和0.37 ± 0.03 [2]。

Temsirolimus能够以剂量依赖的方式抑制PC-3肿瘤的生长，并且对DU145肿瘤的生长抑制作用更大。经Temsirolimus（20 mg/kg i.p.）治疗5次每周，为期3周后，使肿瘤体积增长至500 mm³的平均延迟时间分别为39 ± 5天和17 ± 3天。小鼠随机接受Temsirolimus（5至10 mg/kg/d）或单纯溶媒作为对照组的治疗[2]，每周治疗6天。在所有测试样本中均观察到疾病负担的显著改善。来自3位患者（96、240和359）的异种移植小鼠在脾脏疾病体积上有统计学上显著的减少[3]。以10次腹腔注射的方式给予Temsirolimus，对8226、OPM-2和U266细胞系的皮下生长诱导了显著的剂量依赖性抗肿瘤反应。Temsirolimus的有效剂量与适度的毒性相关，仅引起短暂的血小板减少和白细胞减少。尽管在8226和OPM-2肿瘤结节中Temsirolimus对p70 mTOR底物的抑制作用相同，OPM-2肿瘤生长对于增殖、血管生成和凋亡诱导的敏感性明显更高[4]。

The Flag-tagged wild-type human mTOR (Flag-mTOR) and Flag-mTOR-SI (S2035I, created by site-directed mutagenesis) DNA constructs were transiently transfected into HEK293 cells. Protein extraction and purification of Flag-mTOR and Flag-mTOR-SI were carried out 48 h later as described previously. In vitro kinase assays of purified Flag-mTOR and Flag-mTOR-SI without or with FKBP12 were done and detected by dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) as described [1].

For all experiments, cells were plated in six-well plates in complete growth media for overnight. Various doses of CCI-779 or rapamycin analogs were added alone or with FK506 for indicated periods of time. Total cellular lysates were prepared using NuPAGE-LDS sample buffer. Equal amounts of proteins were subjected to immunoblotting analysis using NuPAGE electrophoresis system. Immunoblots were probed with appropriate primary and secondary antibodies following the manufacturer's instructions and detected using enhanced chemiluminescence. β-Actin immunoblotting and/or reversible Ponceau-S staining of nitrocellulose membranes after the transfer was used to visualize the total protein loading [1].

Mice bearing PC-3 tumors were treated with CCI-779 (1, 5, 10, and 20 mg per kg per day), or vehicle solution for 3 or 5 days per week for 3 weeks. Mice bearing DU145 tumors were only treated with CCI-779 (20 mg per kg per day) or vehicle solution for 3 weeks. Mice bearing PC-3 tumors received the following treatments: (a) control, vehicle solution for CCI-779; (b) chemotherapy alone, mitoxantrone 1.5 mg/kg or docetaxel 10 mg/kg was injected i.p. weekly for 3 doses; (c) CCI-779 alone, 5 or 10 mg/kg was injected i.p. daily, three times a week for 3 weeks; (4) chemotherapy followed by CCI-779. The largest and perpendicular diameters of tumors were measured twice weekly, and animals were coded using ear tags so that the observer was unaware of their treatment history. Tumor volume was estimated and plotted against time to evaluate response to treatment [2].