Carfilzomib (PR-171) is a proteasome inhibitor that irreversibly binds to the chymotrypsin of the 20S proteasome. Carfilzomib has antitumor activity and may be used to treat multiple myeloma.

Proteasome:5 nM

方法：骨髓瘤细胞系 RPMI 8226 和 ANBL-6 用 Carfilzomib (0-25 nM) 处理 24 h，使用 WST-1 assay 检测细胞活力。
结果：Carfilzomib 处理剂量依赖性降低 RPMI 8226 和 ANBL-6 细胞的细胞活力，IC50 均小于 5 nM。[1]
方法：B 细胞淋巴瘤细胞系 Raji、Raji-2R 和 Raji-4RH 用 Carfilzomib (1-10 nM) 处理 24-72 h，使用 Flow Cytometry 分析细胞凋亡情况。
结果：Carfilzomib 诱导 B 细胞淋巴瘤细胞系发生凋亡，细胞凋亡的程度是与时间相关的。[2]

方法：为检测体内抗肿瘤活性，将 Carfilzomib (5 mg/kg，10% captisol) 静脉注射给携带套细胞淋巴瘤 (MCL) Mino 的 SCID 小鼠，每周两次，持续五周。
结果：Carfilzomib 几乎消除了肿瘤生长，并显著延长了荷瘤小鼠的生存时间。[3]
方法：为检测体内抗肿瘤活性，将 Carfilzomib (6 mg/kg，10% captisol) 腹腔注射给转移性无定形甲状腺肿瘤 (ATC) 小鼠模型，每周三次，持续三周。
结果：Carfilzomib 治疗患有已确定的广泛转移性疾病的小鼠显著提高了它们的存活率，但没有显著毒性。[4]

Enzyme-linked immunosorbent assay for subunit profiling of carfilzomib: ANBL-6 cells (2 × 106/well) are plated in 96-well plates and treated with Carfilzomib doses from 0.001 to 10 μM for 1 hour. Cells are then lysed (20 mM Tris-HCl, 0.5 mM EDTA), and cleared lysates are transferred to polymerase chain reaction (PCR) plates. A standard curve is generated using untreated ANBL-6 cell lysates starting at a concentration of 6 μg protein/μL. The active site probe [biotin-(CH2)4-Leu-Leu-Leu-epoxyketone; 20 μM] is added and incubated at room temperature for 1 hour. Cell lysates are then denatured by adding 1% sodium dodecyl sulfate (SDS) and heating to 100°C, followed by mixing with 20 μL per well streptavidin-sepharose high-performance beads in a 96-well multiscreen DV plate and incubated for 1 hour. These beads are then washed with enzyme-linked immunosorbent assay (ELISA) buffer (PBS, 1% bovine serum albumin, and 0.1% Tween-20), and incubated overnight at 4°C on a plate shaker with antibodies to proteasome subunits. Antibodies used included mouse monoclonal anti-β1, anti-β2, anti-β1i, and anti-β5i, goat polyclonal anti-β2i, and rabbit polyclonal anti-β5 (affinity-purified antiserum against KLH-CWIRVSSDNVADLHDKYS peptide). The beads are washed and incubated for 2 hours with horseradish peroxidase-conjugated secondary goat antirabbit, goat antimouse or rabbit antigoat antibodies. After washing, the beads are developed using the supersignal ELISA picochemiluminescence substrate. Luminescent detection is performed. Raw luminescence is converted to μg/mL by comparison with the standard curve and expressed as the % inhibition relative to vehicle control. Curve fits are generated using the following nonsigmoidal dose-response equation: Y = Bottom + (Top-Bottom)/(1 + 10?((LogEC50 ? X) × HillSlope)), where X is the logarithm of concentration, Y is the % inhibition, and EC50 is the dose showing 50% effect.

WST-1 is used to determine the effects of proteasome inhibitor Carfilzomib on cell proliferation. The inhibition of proliferation is calculated in relation to parallel control cells that receives vehicle alone. A linear spline function is used to interpolate the median inhibitory concentration (IC50) using XLfit 4 software. The degree of resistance (DOR) is calculated using the formula: DOR = IC50(resistant cells)/IC50(sensitive cells). ANBL-6 cells pulsed with 100 nM carfilzomib are washed and suspended in PBS containing 5 μg/mL of JC-1, which exhibits potential-dependent accumulation in mitochondria. Analysis of the mitochondrial membrane potential-dependent color shift from 525 to 590 nm is carried out on a FacScan, and the data are analyzed with CellQuest software.(Only for Reference)