Cyclopamine (11-Deoxojervine)

Alias: HSDB3505; 11-deoxojervine; HSDB 3505; HSDB-3505

Cat No.:V1332 Purity: ≥98%

COA Product Data Instructions for use SDS

Cyclopamine (formerly HSDB3505; HSDB-3505; 11-deoxojervine),a naturally occuring steroid alkaloid found in Veratrum plant, is reported to be a potent and specific inhibitor of Hedgehog (Hh) signaling pathway with both anticancer and teratogenic activities.

Cyclopamine (11-Deoxojervine) Chemical Structure CAS No.: 4449-51-8
Product category: Hedgehog(Smoothened) ROCK

This product is for research use only, not for human use. We do not sell to patients.

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Citations by Top Journals: Nature, Cell, Science, etc.

Top Publications Citing lnvivochem Products

Nature 2021, 597(7874):119-125.

Cell 2020,183:1202-1218.e25.

Science Adv 2022: 8, eabm9427.

Nature 597, 119–125 (2021)

Cell Stem Cell 2020,26(6):845-861.e12.

Science Trans Med 2023,15(701):eadd7872.

STTT 2023,8(1):91.

Cell Reports 2023,42(4):112313

Science Trans Med 2023, 15: eadd7872.

Cancer Cell 2021,39(4):529-547.e7.

Cancer Discov 2022,12(4):1106-1127.

Immunity 2023,56(3):620-634.e11.

J Am Chem Soc 2022,144:21831-21836.

Cell 2020,183:1202-1218.e25.

Science Adv 2022:8,eabm9427.

Nature 2021,597(7874):119-125.

Cell 2020,183:1202-1218.e25.

PNAS Nexus 2022,1(3):pgac063.

ACS Nano 2023,17(10):9110-9125.

Biomaterial 2023 Sep16:302:122333.

Purity & Quality Control Documentation

Current batch：

Purity: ≥98%

COA

MSDS

NMR

Instructions

Product Description
Bioactivity & Protocols
Physicochemical Properties
Solubility Data
Calculators
Biological Data

Product Description

Cyclopamine (formerly HSDB3505; HSDB-3505; 11-deoxojervine), a naturally occuring steroid alkaloid found in Veratrum plant, is reported to be a potent and specific inhibitor of Hedgehog (Hh) signaling pathway with both anticancer and teratogenic activities. In TM3Hh12 cells, it inhibits Smoothened (Smo) at an IC50 of 46 nM.

Biological Activity I Assay Protocols (From Reference)

Targets

Human Endogenous Metabolite

ln Vitro

In vitro activity: Cyclopamine blocks the activity of the human Smo receptor expressed in CHO-K1 cells in the [³H]Hh-Ag binding assay with an IC50 of 280 nM and inhibits the Hedgehog signaling pathway with an IC50 of 46 nM.[1] Cyclopamine suppresses the Hedgehog pathway activity in gut-derived tumor cell lines that express Patched (PTCH) mRNA in a dose-dependent manner, and causes growth inhibition of those tumor cell lines by 75-95% at 3 μM. However, it is ineffective against colon tumor cells that do not express PTCH mRNA, indicating that the effects of cyclopamine treatment are related to the Hedgehog pathway rather than being generally cytotoxic.[2] Cyclopamine (10 μM) inhibits the proliferation of SMO^high Cyclopamine-responsive cell lines L3.6sl and Panc 05.04 by 75–80% and increases apoptosis by 2.5–3.5 times, while having no effect on the BxPC3-SMO^low cell line. This is achieved by blocking Hedgehog signaling through direct interaction with Smo.[3] In the E3LZ10.7 cell line, cyclopamine treatment dramatically reduces Snail mRNA and increases E-cadherin transcripts. Cyclopamine treatment significantly inhibits the invasive phenotype of Hedgehog-dependent L3.6pl cells, causing a >500-fold reduction in the number of transmigrating cells, independent of inhibition of cell growth. However, this effect is not observed for the Hedgehog-independent cell line Panc-1.[4]

ln Vivo

In mice, HUCCT1 xenografts are eliminated after 22 days of administration of cyclopamine at a dose of 50 mg/kg/day with no discernible side effects.[2] Cyclopamine treatment at a dose of 1.2 mg for 7 days causes a significant amount of tumor cell apoptosis and reduces the tumor mass by 50–60% in tumors derived from Panc 05.04 and L3.6sl, respectively, but not in tumors derived from BxPC3-SMO^low.[3] Cyclopamine administration by itself significantly reduces tumor metastases in xenografts of E3LZ10.7 and L3.6pl, and when combined with gemcitabine, completely eliminates metastases.[4]

Enzyme Assay

Using Luciferase as a readout, the Gli-Luc assay measures the transcriptional modulation of Gli, the final stage of the Hh signaling pathway. After serial dilution in DMSO, cyclopamine is ready for assay and added to assay plates that are empty. After being resuspended in F12 Ham's/DMEM (1:1) containing 5% FBS and 15 mM Hepes pH 7.3, TM3Hh12 cells (TM3 cells with the Hh-responsive reporter gene construct pTA-8xGli-Luc) are added to assay plates and incubated with Cyclopamine for about 30 minutes at 37°C in 5% CO₂. After that, assay plates are filled with 1 nM Hh-Ag 1.5 and allowed to incubate at 37 °C with 5% CO₂. Following 48 hours, the assay plates are refilled with either Bright-Glo or MTS reagent, and the absorbance or luminescence at 492 nm is measured. The logistic curve's inflection point, or IC50 value, is found by non-linearly regressing the Gli-driven luciferase luminescence or absorbance signal from the MTS assay against log10 (cyclopamine concentration) using the R statistical software package.

Cell Assay

In 96-well plates, cells are exposed to cyclopamine. Soluble tetrazolium salt, or MTS, assay is used to measure cell viability. Using the CellTiter96 colorimetric assay, optical density measurements at 490 nm (OD₄₉₀) at 2 and 4 days determine the viable cell mass. The formula for calculating relative growth is OD (day 4)⋣OD (day 2)/OD (day 2).

Animal Protocol

Mice: Subcutaneous injections of 0.1 mL Hanks balanced salt solution and matrigel (1:1) containing 2×10⁶ cells are administered to CD-1 nude mice. All subjects receive treatment at the same time after the tumors are grown for four days to a minimum volume of 125 mm³. Mice receive subcutaneous injections of either a vector (triolein:ethanol 4:1 v/v) or a suspension of cyclopamine (1.2 mg per mouse in triolein:ethanol 4:1 v/v) every day for seven days. Tumors removed from mice at the conclusion of treatment are weighed, fixed for three hours at 4°C using 4% paraformaldehyde, embedded in paraffin wax, and sectioned (6 µm). Apoptotic cells are detected with recombinant Tdt via TUNEL. Eosin is then used as a counterstain on the sections. Random selection is used to select eight ×20-magnified fields from regions representing the outside, middle, and inside of two control and two cyclopamine-treated tumors. The quantity of TUNEL-positive nuclei was manually tallied. Staining with hematoxylin and eosin is done.

References

[1]. Bioorg Med Chem Lett . 2009 Jan 15;19(2):328-31.

[2]. Nature . 2003 Oct 23;425(6960):846-51.

[3]. Nature . 2003 Oct 23;425(6960):851-6.

[4]. Cancer Res . 2007 Mar 1;67(5):2187-96.

These protocols are for reference only. InvivoChem does not independently validate these methods.

Physicochemical Properties

Molecular Formula

C27H41NO2

Molecular Weight

411.62

Exact Mass

411.31

Elemental Analysis

C, 78.78; H, 10.04; N, 3.40; O, 7.77

CAS #

4449-51-8

Related CAS #

4449-51-8

Appearance

Solid powder

SMILES

C[C@H]1C[C@@H]2[C@H]([C@H]([C@]3(O2)CC[C@H]4[C@@H]5CC=C6C[C@H](CC[C@@]6([C@H]5CC4=C3C)C)O)C)NC1

InChi Key

QASFUMOKHFSJGL-LAFRSMQTSA-N

InChi Code

InChI=1S/C27H41NO2/c1-15-11-24-25(28-14-15)17(3)27(30-24)10-8-20-21-6-5-18-12-19(29)7-9-26(18,4)23(21)13-22(20)16(27)2/h5,15,17,19-21,23-25,28-29H,6-14H2,1-4H3/t15-,17+,19-,20-,21-,23-,24+,25-,26-,27-/m0/s1

Chemical Name

(3S,3'R,3'aS,6'S,6aS,6bS,7'aR,9R,11aS,11bR)-3',6',10,11b-tetramethylspiro[2,3,4,6,6a,6b,7,8,11,11a-decahydro-1H-benzo[a]fluorene-9,2'-3a,4,5,6,7,7a-hexahydro-3H-furo[3,2-b]pyridine]-3-ol

Synonyms

HSDB3505; 11-deoxojervine; HSDB 3505; HSDB-3505

HS Tariff Code

2934.99.9001

Storage

Powder -20°C 3 years

4°C 2 years

In solvent -80°C 6 months

-20°C 1 month

Shipping Condition

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility Data

Solubility (In Vitro)

DMSO: <1 mg/mL

Water: <1 mg/mL

Ethanol: 2~20 mg/mL (4.9~48.6 mM)

Solubility (In Vivo)

10% DMSO+30% PEG 300+5% Tween 80+ddH2O: 1mg/mL

(Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.4294 mL	12.1471 mL	24.2943 mL
	5 mM	0.4859 mL	2.4294 mL	4.8589 mL
	10 mM	0.2429 mL	1.2147 mL	2.4294 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.

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In vivo Formulation Calculator (Clear solution)

Step 1: Enter information below (Recommended: An additional animal to make allowance for loss during the experiment)

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Calculation results

Working concentration： mg/mL；

Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.

Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH₂O，mix and clarify.

Note： (1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.

Biological Data

A, viable cell masses, determined using MTT assays, were reduced by nearly 100% to 0% by Hh inhibition with 6 μmol/L cyclopamine for 4 d as compared with solvent-treated cells in vitro. Cancer Res . 2007 Mar 1;67(5):2187-96.

A, using the Guava Multicaspase assay, an increase of cells undergoing apoptosis was detected on treatment with cyclopamine. Cancer Res . 2007 Mar 1;67(5):2187-96.
Effects of cyclopamine treatment on pancreatic adenocarcinoma cells. Nature . 2003 Oct 23;425(6960):851-6.
Cyclopamine treatment blocks tumour formation of human pancreatic adenocarcinoma cells after transplantation into nude mice. Nature . 2003 Oct 23;425(6960):851-6.