Cyclopamine (11-Deoxojervine), a Smoothened (Smo) antagonist (IC50: 46 nM in TM3Hh12 cells), belongs to the group of steroidal jerveratrum alkaloids.

Smo:46 nM (TM3Hh12 cells)

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]。

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]。

This assay measures the end stage of the Hh signaling pathway, that is, the transcriptional modulation of Gli, using Luciferase as readout (Gli-Luc assay). Cyclopamine is prepared for assay by serial dilution in DMSO and then added to empty assay plates. TM3Hh12 cells (TM3 cells containing Hh-responsive reporter gene construct pTA-8xGli-Luc) are resuspended in F12 Ham's/DMEM (1:1) containing 5% FBS and 15 mM Hepes pH 7.3, added to assay plates and incubated with Cyclopamine for approximately 30 minutes at 37 °C in 5% CO2. 1 nM Hh-Ag 1.5 is then added to assay plates and incubated at 37 °C in the presence of 5% CO2. After 48 hours, either Bright-Glo or MTS reagent is added to the assay plates and luminescence or absorbance at 492 nm is determined. IC50 value, defined as the inflection point of the logistic curve, is determined by non-linear regression of the Gli-driven luciferase luminescence or absorbance signal from MTS assay vs log10 (concentration) of Cyclopamine using the R statistical software pack [1].

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].