Cyclopamine

Smo:46 nM (TM3Hh12 cells)

为了考察Cyclopamine治疗在活体内的效果，首先在无胸腺小鼠中建立了来自HUCCT1细胞的皮下异种移植物，这些细胞来自于具有转移性的胆管癌细胞系。在Cyclopamine处理的动物中，肿瘤在12天内完全消退[3]。在延迟治疗模型中，对比未治疗的对照组，使用Cyclopamine(1.2 mg)处理的BxPC3-SMOlow肿瘤在重量上没有差异。相反，Panc 05.04和L3.6sl来源的肿瘤质量分别观察到了50-60%的减少（见图5b, c）—并且，在同时治疗模型中，L3.6sl来源的肿瘤质量减少了84%，显示了更显著的效果[5]。

将鸡胚胎暴露于cyclopamine后，观察到显著的外部缺陷，包括单眼、小眼、象鼻形成、无肢、胸椎前凸和体积减小[2]。与tomatidine对照组相比，cyclopamine处理使来自食道、胃、胆道和胰腺的肿瘤细胞系的生长降低了75-95%[3]。在胰腺癌细胞系中，使用cyclopamine进行Hh抑制，导致snail的下调和E-细胞黏附分子的上调，与上皮-间质转变的抑制一致，并且通过体外侵袭能力的显著降低得到反映(P < 0.0001)[4]。

DMSO:4.12 mg/mL (10 mM)

Cells were cultured in triplicate in 96-well plates in assay media to which 5E1 monoclonal antibody, ShhNp and/or cyclopamine were added at 0 h at concentrations indicated in the main text. Viable cell mass was determined by optical density measurements at 490 nm (OD490) at 2 and 4 days using the CellTiter96 colorimetric assay. Relative growth was calculated as OD (day 4) 2 OD (day 2)/OD (day 2) [3].

A total of 0.1 ml Hanks' balanced salt solution and matrigel (1:1) containing 2 × 10^6 cells were injected subcutaneously into CD-1 nude mice. Tumours were grown for 4 days to a minimum volume of 125 mm3; treatment was initiated simultaneously for all subjects. Mice were injected subcutaneously with vector alone (triolein:ethanol 4:1 v/v) or a cyclopamine suspension (1.2 mg per mouse in triolein: ethanol 4:1 v/v) daily for 7 days. At the end of the treatment period, tumours were excised from mice, weighed and then fixed for 3 h at 4 °C with 4% paraformaldehyde, embedded in paraffin wax and sectioned (6 μm). Apoptotic cells were identified by TUNEL using recombinant Tdt as previously described29. Sections were then counterstained with eosin. Eight ×20-magnified fields from regions corresponding to the exterior, middle and interior of two control and two cyclopamine-treated tumours were chosen at random [5].