Chloroquine phosphate (Aralen phosphate) is an aminoquinoline antimalarial and also is widely used as an autophagy inhibitor. Chloroquine also is an inhibitor of toll-like receptors (TLRs).

Cytotoxicity against human K562 cells:31.83 nM, Cytotoxicity against hμMan KB cells by microplate method:0.6 μM

Chloroquine （20 μM）可抑制 IL-12p70 的释放，并降低活化的人单核细胞衍生的朗格汉斯样细胞（MoLC）的 Th1 诱导能力。Chloroquine （20 μM）可增强 IL-1 诱导的 MoLC IL-23 分泌，并随后增加启动的 CD4+ T 细胞的 IL-17A 释放[1]。Chloroquine （25 μM）可抑制亲代 MDA-MB-231 细胞在常氧和缺氧情况下的 MMP-9 mRNA 表达。Chloroquine 对 MMP-2、MMP-9 和 MMP-13 mRNA 的表达具有细胞、剂量和缺氧依赖性影响[2]。使用Chloroquine 抑制 TLR7 和 TLR9 可显著减少体外 HuH7 细胞的增殖[3]。

在正位小鼠模型中，Chloroquine （80 mg/kg，静脉注射）不能阻止 TLR9 表达水平高或低的三阴性 MDA-MB-231 细胞的生长[2]。使用Chloroquine 抑制 TLR7 和 TLR9 可显著抑制小鼠异种移植模型中肿瘤的生长。Chloroquine 也能显著抑制 DEN/NMOR 大鼠模型中 HCC 的发展 [3]。

The cells are cultured in 6-well plates with normal culture medium in the presence of vehicle or 25 or 50 μM chloroquine, until near confluency, after which they are rinsed with sterile phosphate-buffered saline (PBS) and cultured further for the indicated times in serum-free culture medium. At the desired time-points, the culture medium is discarded and the cells are quickly harvested in lysis buffer and clarified by centrifugation. Subsequent to boiling the supernatants in reducing sodium dodecyl sulfate (SDS) sample buffer, equal amounts of protein (100 μg) are loaded per lane and the samples are electrophoresed into 10 or 4-20% gradient polyacrylamide SDS gels, then transferred to a nitrocellulose membrane. To detect TLR9, the blots were incubated overnight at 4°C with anti-TLR9 antibodies, diluted 1:500 in Tris-buffered saline with 0.1% (v/v) Tween-20 (TBST). Equal loading is confirmed with polyclonal rabbit anti-actin. Secondary detection is performed with horseradish peroxidase-linked secondary antibodies. The protein bands are visualized by chemiluminescence using an ECL kit [2].

Control and TLR9 siRNA MDA-MB-231 cells (5×105 cells in 100 μL) are inoculated into the mammary fat pads of four-week-old, immune-deficient mice (athymic nude/nu Foxn1). Treatments are started seven days after tumor cell inoculation. The mice are treated daily either with intraperitoneal (i.p.) chloroquine (80 mg/kg) or vehicle (PBS). The animals are monitored daily for clinical signs. Tumor measurements are performed twice a week and tumor volume is calculated according to the formula V=(π/6) (d1×d2)3/2, where d1 and d2 are perpendicular tumor diameters. The tumors are allowed to grow for 22 days, at which point the mice are sacrificed and the tumors are dissected for a final measurement. Throughout the experiments, the animals are maintained under controlled pathogen-free environmental conditions (20-21°C, 30-60% relative humidity and a 12-h lighting cycle). The mice are fed with small-animal food pellets and supplied with sterile water ad libitum [2].