BIJLSMA, Maarten, Fokke (Tilanusstraat 34-II, BK Amsterdam, NL-1091, NL)
SPEK, Christoffel, Amoldus (F. Bordewijkstraat 8, BP Almere, NL-1321, NL)
PEPPELENBOSCH, Maikel, Petrus (Obrechtstraat 74, EH Utrecht, NL-3572, NL)
BIJLSMA, Maarten, Fokke (Tilanusstraat 34-II, BK Amsterdam, NL-1091, NL)
SPEK, Christoffel, Amoldus (F. Bordewijkstraat 8, BP Almere, NL-1321, NL)
C L A I M S
1. A pharmaceutical composition comprising an inhibitor of the enzyme 7-dehydrocholesterol reductase (DHCR 7 ) together with a pharmaceutically acceptable carrier or excipient.
2. The pharmaceutical composition according to claim 1, wherein the inhibitor is tzans-1, A-bis (2-chlorobenzylammomethylcyclohexane (AY- 9944) or a pharmaceutically acceptable salt thereof.
3. The pharmaceutical composition according to claim 1 or 2, wherein the composition also comprises at least one compound chosen from vitamin D3, hydroxylated vitamin D3, vitamin D2 or 7- dehydrocholesterol .
4. A method of preparing a pharmaceutical composition suitable for the treatment of an adenocarcinoma, the method involving a step of including an inhibitor of the enzyme 7-dehydrocholesterol reductase (D 7 reductase) .
5. The method according to claim 4, wherein the inhibitor is trans- 1, 4-jbzs (2-chlorobenzylammomethylcyclohexane (AY-9944) or a pharmaceutically acceptable salt thereof.
6. The method according to claim 4 or 5, wherein the method involves a step of including vitamin D3, hydroxylated vitamin D3 , vitamin D2 or 7-dehydrocholesterol m the pharmaceutical composition.
7. The method according to any of the claims 4 to 6, wherein the adenocarcinoma is chosen from the group consisting of prostate cancer, breast cancer, medulloblastoma, basal cell carcinoma and small cell lung cancer, and preferably the adenocarcinoma is an adenocarcinoma from the upper gastrointestinal tract and m particular it is chosen from the group consisting of hepatic, oesophageal, gastric and pancreatic adenocarcinoma. |
A pharmaceutical composition suitable for the treatment of cancer and method of preparing said pharmaceutical composition
The present invention relates to a pharmaceutical composition suitable for the treatment of cancer.
Despite years of medical research, cancer is still a prevalent disease. While progress has been made for some types of cancer, such as melanoma, it is well known that for other types of cancer, in particular adenocarcinomas, no suitable treatment is available or alter- native treatments would be desirable for non-responsive cancers or for combination therapy.
It is the object of the present invention to provide a pharmaceutical composition for the treatment of cancer, in particular but not limited to adenocarcinomas of the upper gastro-intestinal tract. To this end, the pharmaceutical composition is characterized in that it comprises an inhibitor of the enzyme 7-dehydrocholesterol reductase (DHCR 7 ) together with a pharmaceutically acceptable carrier or excipient.
Research by the present inventors has revealed that vitamin D3 plays an important role in the Hedgehog pathway. Hedgehog is a protein that plays an important role in embryonic development, and is a morphogen, i.e. an agent that provides spatial information via a concentration gradient during embryonic development. The Hedgehog pathway involves two receptors, known as Patched and Smoothened. Once a Hedgehog protein (a member of a class of proteins capable of undergoing autocatalysis) binds to Patched, Patched no longer inhibits Smoothened. As a result, the Smoothened receptor becomes active, resulting in Smoothened-mediated gene expression. Excessive Smoothened- mediated gene expression is associated with various cancers. By blocking the degradation of (pro) vitamin D3 in accordance with the teaching of the present invention, this vitamin is accumulated, as a result of which Smoothened is blocked. The experimental section of the present application contains the proof of the physiological role of (pro) vitamin D3. In addition, as evidence is provided that block- ing "of the degradation of vitamin D3 using an inhibitor of an enzyme involved in the degradation of vitamin D3 results in the death of adenocarcinoma cells. For the sake of accuracy, it is remarked that it is generally recognized that the term "degradation of vitamin D3"
not necessarily means that vitamin D3 itself serves as a substrate for an enzyme, and that a conversion of vitamin D3 can occur, which conversion product (in particular 7-dehydrocholesterol) is the actual product that is degraded. The involvement of such an intermediate step is within the meaning of the term "degradation of vitamin D3" as used in the present application.
According to a preferred embodiment, the inhibitor is trans- 1, 4-jbis (2-chlorobenzylaminomethylcyclohexane (AY-9944) or a pharmaceutically acceptable salt thereof. This inhibitor appears to be effective at killing cells of adenocarcinomas .
According to a preferred embodiment, the composition also comprises at least one compound chosen from vitamin D3, hydroxylated vitamin D3, vitamin D2 or 7-dehydrocholesterol. These compounds inhibit Smoothened directly.
The invention also relates to a method of preparing a pharmaceutical composition suitable for the treatment of an adenocarcinoma, the method involving a step of including an inhibitor of the enzyme 7-dehydrocholesterol reductase (DHCR 7 ) . Including this compound in the pharmaceutical composition provides for a drug useful to combat cancer. The present invention also relates to the use of an inhibitor of the enzyme 7-dehydrocholesterol reductase (DHCR 7 ) for the preparation of a pharmaceutical composition suitable for the treatment of an adenocarcinoma. An effective inhibitor is trans-1, 4-bis (2-chlorobenzyl- aminomethylcyclohexane (AY-9944) or a pharmaceutically acceptable salt thereof.
For the reason already cited above, according to a preferred embodiment, the method involves a step of including vitamin D3, hy- droxylated vitamin D3 , vitamin D2 or 7-dehydrocholesterol in the pharmaceutical composition.
According to a preferred embodiment, the adenocarcinoma is chosen from the group consisting of prostate cancer, breast cancer, medulloblastoma, basal cell carcinoma and small cell lung cancer, and more 'preferably the adenocarcinoma is an adenocarcinoma from the up- per--,"gastrointestinal tract and in particular it is chosen from the group consisting of hepatic, oesophageal, gastric and pancreatic adenocarcinoma.
The present invention also relates to a method of treating a patient suffering from an adenocarcinoma, wherein the patient is treated with a pharmaceutical composition according to the invention or a pharmaceutical composition prepared using the method according to the invention, as described above including the preferred embodiments thereof.
The invention also relates to a method of preparing a pharmaceutical composition suitable for the treatment of an adenocarcinoma, the method involving a step of including vitamin D3, hydroxylated vi- tamin D3 , vitamin D2 or 7-dehydrocholesterol in the composition.
In this method, the active ingredient is preferably prepared as a suspension, or as liposomes.
As oral administration might not result in high enough concentrations of vitamin D3 in the target tissue due to its hydrophobic- ity, injection is the preferred method of administration, albeit sys- temically or locally. The latter is preferred as high systemic concentrations of vitamin D3 might cause side effects such as hypercalcemia .
The invention will now be elucidated with reference to the following experiments and with reference to the drawings. For each of the figures 1 - 9 an extensive legend is provided at the end of the experimental section.
In section 1 rigorous proof is provided for the inhibitory role of Vitamin D3 m the Hedgehog pathway, and in section 2 expeπ- ments are shown demonstrating the effect of an inhibitor that causes vitamin D3 levels to rise and concomitantly cancer cell mortality.
Section 1
Intracellular signal transduction of the morphogen Hedgehog (Hh) is remarkable, with many features exclusive to this signalling system, many of which are only partly understood (Bi]lsma et al . , 2004). Hh signalling is initiated by two membrane proteins, Patched (Ptchl) and Smoothened (Smo) , the former a 12-pass transmembrane protein (Ingham et al . , 1991; Marigo et al . , 1996) resembling the Nie- mann-Pick disease type Cl protein (which is involved m cholesterol trafficking and which has a pump function), the latter a 7-pass transmembrane protein resembling a G protein-coupled receptor (van den Heuvel and Ingham, 1996) . In the absence of the inhibitory recep-
tor Ptchl, Smo is constitutively active and leads to the activation and nuclear translocation of its downstream transcription factor glioma-associated (GIi) . In normal physiology, activation of the signalling pathway is caused by binding of Hh to Ptchl, resulting in the internalization of Ptchl and consequently alleviation of the inhibitory effect of Ptchl on Smo (Denef et al . , 2000; Incardona et al . , 2002) . The exact mechanism behind Ptchl inhibition of Smo however, remains unclear (Hooper and Scott, 2005) . Here we report the mechanism by which Ptchl inhibits Smo. Similarities m the phenotypes of humans with inherited disorders of sterol biosynthesis (as depicted in Figure IA, lathosterolo- sis and SLOS) and the phenotypes seen with mutations m the Hh signalling pathway have led to the suggestion that cholesterol synthesizing enzymes may somehow be involved in Ptchl dependent Smo lnhibi- tion (Bijlsma et al., 2004). Figure IB depicts the proposed possible modes of Ptchl action on Smo. As Ptchl and Smo do not unambiguously show physical association, and Ptchl can inhibit Smo substoichiomet- πcally, direct binding of Ptchl to Smo as shown in Figures IB-I and 1B-2 seems an unlikely mechanism of inhibition (Taipale et al . , 2002) . As mentioned, Ptchl shares high homology (like for instance a sterol sensing domain) with NPCl (Davies et al . , 2000), and with various (prokaryotic) pump proteins. Also, several small molecules with homology to cholesterol that act as Hh pathway antagonists have been identified (Frank-Kamenetsky et al . , 2002; Incardona et al., 1998; Taipale et al., 2000) . For instance, cyclopamme, a well-known antagonist of Smo, has been widely used for assessing the effects of Hh pathway inhibition in several model systems (e.g (van den Brink et al., 2004)). Consequently, it has been anticipated that m the absence of Hh, Ptchl translocates a small molecule resembling choles- terol across the membrane, acting as a Smo antagonist and this hypothesis is summarized in Figure 1B-3. We set out to test this prediction and have now surprisingly established that vitamin D3 is the naturally occurring inhibitory ligand of Smo. Hh thus activates its signalling cascade by inhibiting Ptchl-dependent secretion of the 3- beta-hydroxysteroid (pro-) vitamin D3.
Validation of a model system to study Ptc-Smo interaction
An experimental system to study intercellular inhibitory actions of Ptchl on Smo should fulfil four requirements: cells must be capable of sustaining Ptchl expression, expressed Ptchl must be functionally active, the inhibition of GIi activity should be Smo- mediated and endogenous Hh should not be a contributing factor. To set up a model system for studying Ptchl dependent inhibition, C3H/10T1/2 fibroblasts (from American Type Culture Collection CCL- 226, Manassas, VA) were transfected with an 8 χ Gli-binding site Iu- ciferase construct (this Gli-reporter d51-LucII was kindly provided by Dr. H. Sasaki as well as the mutant binding site variant (mGli) (Sasaki et al . , 1999)) together with Ptchl, Smo, Smo and Ptchl or Glil. The use of the luciferase construct enables us to monitor the transcriptional activity of the Hh pathway.
Mouse Ptchl (a generous gift of Dr. M. P. Scott) was cloned into the pcDNA 3.1(-) vector (Invitrogen, Carlsbad, California). Human SMO (referred to as Smo) cDNA (Image Consortium/RZPD, Berlin, Germany) was cloned into the pcDNA 3.1(+) vector. The Glil cDNA is in pcDNAl (a generous gift of Dr. A. Ruiz i Altaba) . The internal control CMV driven Renilla luciferase vector was from Promega (Madison, WI). SmoM2 in pRK7 was obtained from Genentech (South San Francisco, CA) . Mouse mesenchymal fibroblasts (C3H/10T1/2) were grown in DMEM (Cambrex, East Rutherford, NJ) containing 10% fetal calf serum (FCS (Cambrex, East Rutherford, NJ) ) . Cells were grown to approximately 70 % confluence and transfected with either Ptchl or Smo and the GIi- luciferase reporter in combination with a CMV-Renilla luciferase internal standard using Effectene (Qiagen, Hilden, Germany) according to standard procedures. Ptchl and scrambled control siRNA were transfected using RNAiFect (Qiagen) following the supplied protocol. The Ptchl (#63908) and scrambled control siRNA were from Ambion (Austin, TX) . Overexpression of Ptchl drove cells into apoptosis (Thibert et al . , 2003) . To overcome this problem, we performed experiments in the presence of 20 μM caspase inhibitor zVADfmk (Sigma-Aldrich, St.- Louis, United States) . Under these conditions, transfection with a Ptchl expression construct led to efficient overexpression of Ptchl as detected by Western blot (Figure 2A) . To assay protein content, transfected cells were lysed in Laemmli buffer and brought onto SDS- PAGE gels. After electrophoresis, protein was transferred onto Immo- bilon-PVDF membranes (Millipore, Billerica, MA) . Membranes were
blocked in 5% BSA (Sigma-Aldrich, St Louis, MO) in TBS/0.1% Tween-20 (TBST) for Ih. Goat polyclonal α-Ptchl antibody G-19 was diluted to 1:500 in 3% BSA in TBST and membranes were incubated overnight. Goat polyclonal α-actin 1-19 (Santa Cruz Biotechnology) was diluted to 1:1000 in 3% BSA in TBST. After Ih incubation in 1:1000 α-goat HRP- conjugated secondary antibody (DakoCytomation, Glostrup, Denmark), blots were imaged using LumiLight Plus ECL (Roche, Basel, Switzerland) on a GeneGnome chemiluminescence imager (Syngene, Cambridge, UK) . Transfection of Ptchl effectively inhibited transactivation of the Gli-reporter in the presence of overexpressed Smo (Figure 2B) . This activation seen by expression of Smo and the inhibition by Ptchl correspond with the known actions of these proteins, which confirms the validity of the construct used. The Ptchl-insensitive mutant SmoM2 (Taipale et al . , 2000; Xie et al . , 1998) was not inhibited by Ptchl cotransfection and showed a high basal GIi activity. This confirms the specificity of Ptchl action on Smo. To assay Hh pathway activity, cells were lysed with passive lysis buffer as provided by Promega and luciferase activity was assayed according to the Promega Dual-Glo Luciferase Assay System (Promega) protocol on a Lumat Ber- thold LB 9501 Luminometer (Berthold Technologies, Bad Wildbad, Germany) . Each Firefly luciferase value (that represents a certain amount of Hh pathway activity) was corrected for its co-transfected CMV driven Renilla luciferase standard to correct for transfection efficiency or dilution effects. The inhibitory effect of the basal Ptchl levels could be overcome by the addition of 1 μg/ml recombinant N-terminal Shh for 6 h, as can be seen from the high reporter activity upon stimulation (Figure 2B) . This confirms that the low levels of GIi activity are due to endogenous Ptchl expression. Addition of 1 μg/ml 5El Shh-blocking antibody (Ericson et al . , 1996) could counteract the stimulation by Shh. The 5El antibody binds to Shh in such a way that Shh is unable to bind to Ptchl, rendering it inactive. This confirms that a high basal level of Ptchl activity is present in the cells used. Recombinant N-terminal Shh was obtained from R&D Systems (Minneapolis, MN) and dissolved in PBS with 0.1% BSA. The 5El Shh- blocking antibody was from the Developmental Hybridoma Bank (Iowa City, IA) . As Smo and GIi overexpression were capable of increasing transactivation of the Gli-reporter, despite the presence of a
caspase inhibitor, our experimental system is a valid readout for Smo-mediated GIi activity.
The requirement for Smo overexpression in order to respond to Ptchl inhibition and the high responsiveness to exogenously added Shh suggests that endogenous Hh production is not a major factor in our setup, although others have shown mRNA for Ihh and Shh in C3H/10T1/2 cells (Shea et al., 2003; Wu et al . , 2004). To exclude Hh protein excretion by our C3H/10T1/2 cells as a contributing factor in our model system we performed Western blot analysis on Shh-spiked medium and medium from fibroblast cell culture. Using the a-Shh N-19 antibody, the cells were not found to excrete Hh protein (Figure 2C) . As can be seen, the detection limit was about 5 ng/ml, which was not reached in the (4 times concentrated) medium, indicating the Hh concentration in the medium to be less than 1.25 ng/ml, a concentration too low to evoke a response (Pepinsky et al . , 1998). Reprobing the blot for Ihh did not yield a signal for the medium as well (a-Ihh 1-19, Santa Cruz Biotechnology, Santa Cruz, CA) . In conclusion, there is no detectable amount of hedgehog protein present in the medium.
Ptchl can inhibit Smo intracellularly
To assess the ability of Ptchl to act upon Smo on another cell, we employed a so-called Mix-and-Match approach (as schematically shown in Figure 3A) , in which two populations of cells are mixed. The donor population of cells expresses Ptchl at different levels (using an overexpression construct or specifically targeted siRNA) that could act on reporter cells with a constitutively active Hh-pathway (reporter cells; Smo receptor overexpressed and a GIi- luciferase reporter) . Cells were washed three times with PBS, detached by 2 mM EDTA (Sigma-Aldrich) m PBS, resuspended in DMEM with 10% FCS and pipetted 3 times through a 40 μm cell strainer (BD Bio- sciences, San Jose, CA) into a tube to allow a homogenous mixture. Equal volumes of reporter and donor cells were mixed thoroughly and subsequently transferred to 24 well plates. After 6h, cells were lysed and Hh pathway activity was assayed. This activity is only measured in the reporter cells and is therefore "uncoupled" from the donor cell population.
We mixed reporter cells with vector, Ptchl, Ptchl siRNA and scrambled control siRNA transfectants . Employing two fluorescent
tracers for labelling the cell populations, the capability of this procedure to obtain a homogenous mixture could be assessed. For fluorescent tracking, cells were grown to approximately 70% confluence in DMEM containing 10% FCS and washed with PBS to remove serum. CeIl- Tracker Green CMFDA or Orange CMTMR (Molecular Probes, Eugene, OR) was diluted to 5 μM in serum-free DMEM and added to cells for 45 minutes, the medium was aspirated and fresh medium containing 10% FCS was added. Cells were detached and mixed as described above. After 16h, cells were fixed in 3.7% formaldehyde overnight or mixed as de- scribed above and subsequently fixed. For fluorescent staining of nuclei, cells were washed with PBS/0.1% Triton X-100 (PBST) and incubated m 200 ng/ml DAPI (Roche, Basel, Switzerland) in PBST for Ih. Prior to microscopy, cells on cover slips were washed briefly in PBS, placed upside-down on another cover slip if imaged on a Leica TCS SP II confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany) or placed on an object glass when imaged on a Zeiss Axioskop (Carl Zeiss Inc., Oberkochen, Germany) . Figure 3A shows that combining two cell populations yields evenly mixed cells with intimate cell-cell contacts (micrograph) . When in these Mix-and-Match expeπ- merits Gli-reporter cells were mixed with Ptchl overexpressmg cells, a significant reduction in Smo-mediated GIi activation could be seen as compared to control. This reduction m GIi activation in the reporter cells is indicative of an intracellular mode of action for Ptchl. This inhibition could not be diminished by addition of 1 μg/ml 5ξ1 SHh-blocking antibody (Figure 3A), again demonstrating that endogenous Hh is not a contributing factor m our experimental set up. This is specifically important as Hh present m the medium would increase reporter activity. As Ptchl scavenges Hh from the medium, medium of Ptchl overexpressmg cells would contain lower Hh levels as control or Ptchl siRNA transfected cells. The reduction m reporter activity as consequence of such scavenging would mask the action of a potential inhibitory molecule. Addition of 1 μg/ml recombinant Shh, which should inhibit Ptchl present in these experiments, was able to abolish the inhibition conferred by Ptchl. Mixing reporter cells with Ptchl siRNA transfected cells increased Smo-dependent reporter activity (Figure 3A) , demonstrating that the inhibition is Ptchl-dependent and again suggests that C3H/10T1/2 cells have a significant basal level of Ptchl activity. Importantly, showing opposite effects of
Ptchl siRNA versus DNA excludes any overexpression artefacts to be responsible for the observed non-cell autonomous effect. To exclude cell-specific artefacts, the procedures were also performed with MDA- MB-231 breast tumour cells ATCC number HTB-26) , which had even higher levels of endogenous PTCH and SMO than the C3H/10T1/2 fibroblasts (Western blot, data not shown) . Indeed, using these cells yielded similar, but more pronounced effects as found with the C3H/10T1/2 cells (Figure 3A, hatched bars) . Human MDA-MB-231 breast carcinoma cells (ATCC number HTB-26) were grown m L-15 Leibovitz medium (Cam- brex) with 10% FCS. These enhanced effects m the presence of higher PTCH and SMO levels demonstrate the specificity of the effects observed .
To determine the maximal inhibition Ptchl confers in a cell autonomous fashion, i.e. the extent to which Ptchl can inhibit Smo on the same cell, we mixed and fused the two previously described cell populations. In a fusion protocol, cell membranes are temporarily disturbed, allowing membranes of different cells to merge. After fluorescent labelling (Ih) or transfection (16h) , cells were mixed, medium was aspirated and cells were washed with PBS. Pre-warmed PEG 1500/Hepes pH7.4 50% (Roche, Basel, Switzerland) was added to the cells for 90 seconds, after which the cells were washed 3 times with PBS. Fresh medium was added and after 2h, cells were either lysed with passive lysis buffer and assayed for luciferase activity, or fixed for microscopy after 8h. PEG1500 induced efficient cell fusion as evident from blended staining of PBG1500-treated cells and the multi-nucleated cells (procedure and micrographs shown an Figure 3B, left panel) . Fusion of reporter cells with Ptchl overexpressing cells again showed a reduction in GIi activation like that observed in the Mix-and-Match experiments (Figure 3A) . Importantly, the reduction in reporter activity was of the same magnitude as that obtained in the mixing experiments. Therefore, we conclude that Ptchl inhibition of GIi- reporter activity in our system is mainly mediated lntercellu- larly. The specificity of the observed inhibitory action of Ptchl acting through Smo was assessed by using the Ptchl-insensitive SmoM2 mutant as previously shown in Figure 2B. As can be seen in Figure 3C, SmoM2 transfected reporter cells are no longer sensitive to mixing with Ptchl overexpressing donor cells, even when co-transfected with wild-type Smo. These findings led us to suggest the model for Ptchl
action as depicted m Figure lB-3 and Figure 3D, m which Ptchl on one cell is capable of specifically inhibiting Smo on another cell; to act non-cell autonomously.
Non-cell autonomous Ptchl-dependent Smo inhibition is carried by a medium-borne factor
The intercellular Ptchl-mediated inhibition of Smo as identified in the Mix-and-Match experiments may be conferred by an inhibitory molecule secreted or translocated by Ptchl. Indeed, proof for our hypothesis that Ptchl secretes a Smo inhibitory molecule into the medium was obtained from experiments in which we conditioned medium on Ptchl -transfected cells that was transferred to reporter cells in which GIi activity was assayed (procedure schematically shown in Figure 4A, left panel) . Donor and reporter cells were transfected for 16h, after which the donor cells were washed extensively to wash out any remaining transfection complexes and supplied with fresh medium for 6h. Medium was transferred to reporter cells for 6-8h after which cells were lysed and assayed for luciferase activity. Using this experimental set-up, medium conditioned on Ptchl-transfected cells was found to exert a strong inhibitory effect on GIi activation as compared to medium conditioned on control transfected cells (Figure 4A) . In other words; the presence of Ptchl adds a compound to the medium that can act on Smo.
This inhibition could not be blocked by addition of 1 μg/ml Shh or 5El to the reporter cells. The inability of Shh to prevent Ptchl-conditioned medium dependent inhibition of Smo activity is expected, as the transferred medium does not contain donor cells expressing Ptchl on which Shh should exert its action, and the presence of the medium-borne factor m the Ptchl-conditioned medium shortcuts the function of endogenous Ptchl in the reporter cells. Using MDA-MB- 231 cells m this set-up, similar results were obtained (hatched bars) . As for the Mix-and-Match experiments, medium conditioned on Ptchl siRNA transfected cells increased Smo-dependent reporter activity in both cell lines. Medium conditioned on mouse embryonic fibro- blasts (MEFs) from Ptchl knockout mice (Goodrich et al., 1997) showed a distinct deficiency m their capacity to inhibit Smo as compared to medium conditioned on wild type {Ptchl+/+) MEF (bars indicated as MEF donor cells) . The mutant mouse embryonic fibroblasts (MEFs) were
grown in complete DMEM supplemented with 15% FCS and non-essential ammo acids (Sigma) . This inhibitory effect could again not be conferred to reporter cells transfected with the Ptchl-insensitive SmoM2. The opposite effects as observed with Ptchl DNA transfection versus either Ptchl siRNA transfection (blue bars) or genetic Ptchl deficient MEFs exclude transfection artefacts on the donor cells as potential cause of the observed effects . Overall, these findings suggest that Ptchl transfers a molecule to the medium that inhibits Smo activity. Using serum-free medium, no inhibitory action of Ptchl- conditioned medium on reporter cells could be conferred (Figure 4A) . As serum-free medium contains no lipoproteins, this implies an important role for lipoproteins in conveying the inhibitory signal from cell to cell. Alternatively, serum-free medium also lacks lipids, which could stress the necessity for lipids supplied in the medium for Hh responsiveness (Cooper et al . , 2003) . However, serum-free conditions for 8 hours, (the incubation time in our experiments) do not deplete a cell's cholesterol metabolism arguing m favour of a role of lipoproteins in transporting the Smo-mhibitory molecule.
Ptchl-dependent secretion of 3β-hydroxysteroids
Media were conditioned on cells transfected with Ptchl or siR- NA for Ptchl and subjected to FPLC-coupled CHOD-PAP analysis, a technique that analyzes 3β-hydroxysteroid content. Using CHOD-PAP-coupled Fast Performance Liquid Chromatography (FPLC) steroid levels in the main lipoprotein classes (VLDL, LDL and HDL) were determined using high performance gel filtration chromatography (HPGC) . The system contained a PU-980 ternary pump with an LG-980-02 linear degasser, FP-920 fluorescence and UV-975 UV/VIS detectors (Jasco, Tokyo, Japan) . An extra P-50 pump (Pharmacia Biotech, Uppsala, Sweden) was used for in-line CHOD-PAP enzymatic reagent (Biomerieux, Marcy l'Etoile, France) addition at 0.1 ml/min. 60 μl from each sample was subjected to size-exclusion chromatography to determine whether there is a relationship between Ptchl expression and medium 3β- hydroxysteroid levels using a Superose 6 HR 10/30 (Pharmacia Biotech) column with Tris-buffered saline (TBS) pH 7.4 at a flow rate of 0.31 ml /mm with inline fluorescence and UV detection on the Jasco system with CHOD-PAP assay described above. Commercially available lipid
plasma standards were used for total cholesterol pattern analysis (SKZL, Nijmegen, The Netherlands) .
The Ptchl conditioned medium was found to be enriched with 3B- hydroxysteroids relative to medium conditioned by control cells (Fig- ures 4B and 4C; quantification of FPLC profiles as itiM total 3β- hydroxysteroid) . Judging from the retention profile, these 3β- hydroxysteroids were present on LDL particles and to a lesser extent on VLDL. HDL 3β-hydroxysteroid levels remained constant in all conditions. Medium incubated on cells transfected with siRNA for Ptchl was virtually devoid of LDL-associated 3β-hydroxysteroids . This correlation between modified Ptchl levels and the accumulation of 3β~ hydroxysteroid molecules in the medium suggests that Ptchl translocates or pumps these 3β-hydroxysteroids into the medium and thereby transfers the Smo inhibitory activity.
3β-hydroxysteroids are necessary for Ptchl-dependent Smo inhibition
In the presence of pravastatin (Sigma-Aldrich) a HMG-CoA reductase inhibitor that abrogates the biosynthesis of the 3β- hydroxysteroid precursor mevalonate (Figure IA) , LDL-associated 3β- hydroxysteroid (named LDL-C on axis) levels were substantially reduced (Figure 4C) and correspondingly, reduced GIi inhibition was observed in Ptchl-overexpressing reporter cells as shown in Figure 4D. Conversely, the 3β-hydroxysteroid precursor mevalonate (Sigma- Aldrich) strongly increased LDL-associated 3β-hydroxysteroids and Ptchl-mediated inhibition of Gli-transactivation. This suggests that a cholesterol like molecule is responsible for the Smo inhibition in the previously mentioned experiments.
If the observed effects of steroid synthesis modifiers on GIi- activation are Smo-specific, Hh responses mediated by Ptchl (but not those by Smo) should remain unaffected. The endocytosis of Ptchl is such a Smo-independent response to Hh (Marigo et al . , 1996; van den Heuvel and Ingham, 1996). Cells on 24-wells plates were grown to 70% confluence and treated with no or 1 mM pravastatin for 6h. Subse- quently, 200 nCi of [3H] -labelled sucrose (Amersham Pharmacia Biotech, Freiburg, Germany) was added per well. Cells were stimulated with either 1 μg/ml Shh or solvent control (0.1% BSA/PBS) for Ih. After washing, cells were lysed in 1% Nonidet P-40 and the lysate was
transferred to 4 ml of scintillation fluid and activity was determined on a Packard Tn-Carb scintillation counter (PerkinElmer, Wellesley, MA) . Values were corrected for solvent control treated cells on ice. Using [3H] -sucrose as a tracer (Peppelenbosch et al . , 1999) we observed no effect of 1 mM pravastatin on Hh-mediated endo- cytosis (Figure 4E) and thus the effects of steroid synthesis inhibitors are Smo-specific . It is important to realize, however, that the above-mentioned reporter data are not from medium transfer experiments and that the inhibition or stimulation of cholesterol synthesis and the concomitant effects on GIi activity do not yet unequivocally prove the contribution of sterol biosynthesis to the intercellular action of Ptchl .
Using Dhcr7-/- and Sc5d-/- MEFs to identify the inhibitory compound
As we hypothesized that 7-DHC might be a Smo inhibitor, we used MEFs (Cooper et al . , 2003) from mice genetically deficient for 7-DHC reductase (Dhcr7-/-) as these cells (obtained from Dr. Denny Porter, Bethesday, MA) stack 7-DHC. To confirm the specificity of 7- DHC as Smo inhibitor, we also employed Sc5d-/~ MEFs that stack the precursor of 7-DHC (lathosterol) and contain little or no 7-DHC. Indeed, as shown in Figure 5A, Dhcr7-/- MEFs had a significantly reduced GIi activity as compared to Sc5d-/- MEFs. In addition, the Smo- inhibitory potential of Dhcrl-/- MEF conditioned medium was much higher then ScSd-/- MEF conditioned medium as shown m the medium transfer experiment depicted in Figure 5A. In addition to stacking a specific metabolite, both MEFs are equally incapable of sterol synthesis. Our data therefore argue against reduced sterol levels as being responsible for the observed Smo inhibition. Overall, these data strongly suggest that 7-DHC or a Dhcr7 independent metabolite of 7- DHC has an inhibitory action on Smo.
To assess whether Ptchl utilizes 7-DHC to inhibit Smo, we performed medium transfer experiments with Ptchl (or Ptchl siRNA) trans- fected Dhcr7-/- and Sc5d-/- MEFs as donor cells. If Ptchl would in- deed pump 7-DHC, Ptchl overexpression or knockdown in the Sc5d-/- MEFs should show no effect on Smo inhibition. As shown m Figure 5B, the Sc5d-/- MEFs were severely hampered in their ability to transfer Ptchl action to the medium, as neither Ptchl DNA nor siRNA transfec-
tants differed from control transfectants in their ability to inhibit Smo on reporter cells. The Dhcr7-/- MEFs, however, were well capable of translating Ptchl expression levels to differential inhibitory action on reporter cells. Interestingly, UVB treatment of Dhcr7-/~ MEF conditioned media, which catalyzes the reaction from 7-DHC to vitamin D3, increased the Ptchl-effect on reporter cells. As UV light catalyzes the conversion of 7-DHC to vitamin D3, we conclude that Ptchl utilizes vitamin D3 to inhibit Smo.
Vitamin D3 is sufficient for Smo inhibition
From the experiments described above, we hypothesized that addition of synthetic 7-DHC or vitamin D3 would inhibit GIi activity in reporter cells as well. Indeed, as can be seen from Figure 6δ, 7-DHC was capable of inhibiting Smo, but was not nearly as potent as its derivative, vitamin D3. This fits the observation that UV treatment enhanced the inhibitory potential of Ptchl conditioned medium (Figure 5B) . Addition of the 7-DHC reductase inhibitor AY-9944 (Repetto et al . , 1990) (Calbiochem, San Diego, CA) successfully enhanced the effect of vitamin D3 treatment, but was also capable of inhibiting Smo by itself, by causing accumulation of endogenously synthesized 7-DHC, or by acting as a 7-DHC mimetic. Cells transfected with the GIi- luciferase reporter were stimulated for the indicated times and concentrations . The magnitude of inhibition conveyed by either the transfer of Ptchl transfectant conditioned medium or Ptchl cotrans- fection were both smaller than that of vitamin D3. In addition, inhibition conferred by vitamin D3 was stronger than that of 10 μM cyclo- pamine (Biomol, Plymouth Meeting, PA) .
Shown in Figure 6B is a dose dependent response of reporter cells to vitamin D3 for 6h. The level of inhibitory N-terminal Gli3 protein increased accordingly, as quantified from Western blot (a- Gli3 N-terminal N-19, Santa Cruz Biotechnology) . This digestion product of Gli3 originates from proteolysis in the SuFu/Fu complex present in Hh-pathway inactive cells, and is considered the repressor form (Ruiz i Altaba, 1999) . To exclude cytotoxic artefacts of vitamin D3 on the C3H/10T1/2 fibroblasts we use, we measured cell viability by MTT reduction. Cells were seeded in flat-bottom 96 wells plates and treated with the indicated concentrations of vitamin D3 for 6h. During the last 3h, 0.5 mg/ml thiazolyl blue tetrazolium bromide
(MTT) was added. After incubation, supernatant was discarded, cells were lysed in 50 ml 40 mM HCl in isopropanol and absorbance was measured at 570 ran in a Benchmark Plus Microplate Spectrophotometer (Bio- Rad, Hercules, CA). Only at very high (1 mM) concentrations of vita- min D3 we could observe a slight decrease in cell viability. Using SmoM2 transfected reporter cells, GIi reporter inhibition occurred only at 100 μM vitamin D3, and below that concentration no inhibition could be observed. This is very similar to the impaired but not absent response of SmoM2 to cyclopamine (Taipale et al., 2000). To exclude Gli-independent effects of vitamin D3 being responsible for the observed reporter inhibition, we used a panel of various luciferase reporter constructs. As can be seen in Figure 6C, in- terferon-stimulated response element (pISRE) , TATA-like promoter (pTAL) , nuclear factor kappa B (pNF-kB) and mutant Gli-binding site (mGli) luciferase reporter constructs were not significantly inhibited by addition of 10 μM vitamin D3 for 6h, whereas reporter activity of a pTAL-luciferase construct was increased. The NF-kB, ISRE and TAL luciferase constructs were from Clontech Laboratories (Mountain- view, CA) . Thus, the inhibitory effect of vitamin D3 on Gli-reporter activity appears to be a genuine effect on Smo-dependent signalling.
Figure 6D shows GIi reporter inhibition by vitamin D3 in the cell lines C3H/10T1/2 and MDA-MB-231 previously employed in the Mix- and-Match and medium transfer experiments. Both showed a marked inhibition in reporter activity following treatment with 10 μM vitamin D3. Importantly, Ptchl -/- MEFs also responded to vitamin D3. As these cells have no Ptchl, any artefacts of vitamin D3 by interference with Ptchl action rather than Smo activity can be excluded.
A consequence of our model in which vitamin D3 or a very similar molecule mediates Ptchl action on Smo (as shown in Figure 6E, summarized in Figure legend) , is that exogenously added vitamin D3 should overrule any effect of Hh on Ptchl. To confirm this, we stimulated stable Gli-luciferase transfectants (Shh-LIGHT II) overnight with 10 μM vitamin D3 and/or 200 ng/ml Shh. What is apparent from Figure 6F, is that in the presence of vitamin D3, reporter activity could not be increased by the addition of Shh, confirming our hypothesis that vitamin D3 plays an essential role in the interaction between Ptchl and Smo.
Scatehard analysis reveals vitamin D3 binding to Smo
To assess possible binding of vitamin D3 to Smo, we used a yeast strain {Pichia pastoris) transformed with Smo of which the expression could be induced by addition of methanol to the growth me- dium (strains obtained from Dr Mus-Veteau, Nice, France; (De Rivoyre et al . , 2005)) . Culture of yeast and induction of Smo expression was performed as described (De Rivoyre et al . , 2005) . We chose this approach for two reasons; first, to our knowledge there is no vitamin D3 receptor in P. pastoris; second, baseline (or background) levels of Smo in non-induced P. pastoris are negligible.
First, we confirmed the successful induction of Smo expression by Western blot using a Smo antibody as shown in Figure 7A (a-Smo C- 17, Santa Cruz Biotechnology). We then set out to perform a Scatehard analysis with heterologous competition by cyclopamine . As cyclopamine is a ligand for Smo, this enabled us to distinguish the observed competition from any background by a-specific binding or specific binding to a vitamin D3 receptor (although not known to exist in these cells) . Scatehard analysis was performed on intact cells as described earlier for (Bloemers et al . , 1998; Pynaert et al . , 1999). After 24h growth in methanol- or glycerol-complex medium, P. pastoris strains were washed with PBS and diluted to the same OD 60O ■ Subsequently, ali- quots of cells were labelled for 1.5h at 4 0 C in PBS containing 1.66 nM of [3H] -labelled vitamin D3 (Amersham Pharmacia Biotech) and 8 different concentrations of unlabelled cyclopamine. The reaction was stopped by washing 4 times with ice-cold PBS. The bound radioactivity was determined by transferring the washed pellets to 4 ml of scintillation fluid and activity was determined on a Packard Tri-Carb scintillation counter (PerkinElmer, Wellesley, MA) . In each experiment each condition was performed in duplicate. In general, Scatehard plots on intact cells show considerable nonspecific low-affinity binding of [3H] -vitamin D3. Therefore Scatehard plots were fitted according to a one or two site model as appropriate. The observed points of non-induced yeast were satisfactory fit with a one-site (low affinity) model, while two affinity sites could be distinguished in the induced P. Pastoris . As can be seen from Figure 7B, [3H]- vitamin D3 was not capable of specific binding to non-methanol induced P. pastoris (BMGY) , whereas after methanol induction (BMMY) specific binding of [3H]- vitamin D3 was observed which was subject
to competition by cyclopamme . Cyclopamme replaced [3H]- vitamin D3 with an apparent Kd of 2 nM in this heterologous assay, a higher affinity than the earlier reported 20 nM affinity of cyclopamme (Chen et al . , 2002) . Nevertheless, these results show that vitamin D3 has the potential to bind Smo at the same site as cyclopamme, indicating that vitamin D3 is indeed a physiological ligand for Smo.
Section 2
Vitamin D3 is cytotoxic m cancer cell lines To determine the cytotoxicity of vitamin D3 for Hedgehog activity dependent cancer cell lines as described for cyclopamme (Berman et al . , 2003; Taipale et al., 2000), we incubated a panel of proximal gastrointestinal tract tumor cell lines in a range of concentrations of vitamin D3 and determined cell viability using the MTT assay as described above. The cells used for these experiments were the HepG2 hepatocellular carcinoma cell line (ATCC number HB-8065) , OE-19 oesophageal adenocarcinoma cells (European Collection of Cell Cultures number 96071721), AGS gastric adenocarcinoma cells (ATCC number CRL-1739) , HMO-2 gastric adenocarcinoma cells (Domhan et al , 2004), BxPC3 pancreatic adenomacarcmoma (ATCC number CRL-1687), and HS 766T pancreatic adenocarcinoma cells (ATCC number HTB-134) , and DLD-I colon cancer cells (ATCC number CCL-221) .
As can be seen in Figure 8A, addition of vitamin D3 for 24h is toxic to all the cancer cell lines tested at 100 μM (compare to Fig- ure 6B, MTT m fibroblasts) , except for the HepG2 hepatocellular carcinoma cells (also described to be sensitive to cyclopamme (Sicklick et al . , 2006) ) . We hypothesize that the rather low sensitivity (high doses of vitamin D3 needed) to vitamin D3 by the fact that these cells are more likely to metabolize vitamin D3 to cholesterol. As previously described, cyclopamme inhibits cell viability in cancer cells that are critically dependent on Hh pathway activity. As we have now found that vitamin D3 acts on the Hh pathway through the same receptor, we set out to compare the potency of cyclopamme and vitamin D3 m reducing tumor cell viability. As can be seen in Figure 8B and C, vitamin D3 is more powerful m reducing cell viability at 100 μM than is cyclopamme. As vitamin D3 is apparently more potent than cyclopamme, is not a known teratogen, and is well tolerated in humans, it offers a promising treatment option.
We predict that the cytotoxicity observed on the presented gastrointestinal cancer cells will also be observed for other cancer types described to be dependent on Hh pathway activity; prostate cancer (Karhadkar et al., 2004), breast cancer (Kubo et al., 2004), me- dulloblastoma (Berman et al . , 2002), basal cell carcinoma (Athar et al . , 2004), small cell lung cancer (Watkins et al . , 2003).
When the inhibitor of the downstream reductase DHCR7 (AY-9944) was added (Figure 8C), no vitamin D3 was needed to reduce cell viability in the AGS gastric adenocarcinoma cell line and the HepG2 cells, and a great toxicity was observed for all cancer cell lines tested. These data confirm the necessity for vitamin D3 metabolism 15 (and thereby avoiding stacking) in order for a cell to survive and indicate the specificity of the effects observed.
To establish the Hh-specificity of vitamin D3 mediated cell death, BXPC3 cells as used in Figure 8 were transfected with control vector, SmoM2 or Glil and subjected to vitamin D3 after which cell viability was determined. The SmoM2 receptor is known not to be sensitive to the established inibitor cyclopamine, and this should thus also exclude vitamin D3 action on cell growth. Overexpression of the downstream transcription factor Glil bypasses any inhibition of Smo by cyclopamine and should also do so for vitamin D3. As can be seen in Figure 9, transfection with either of the two abovementioned constructs strongly diminished vitamin D3 cytotoxicity, indicating its specificity in acting through the Hh pathway. To confirm the notion that Hh inhibition specifically targets tumors from the proximal gastrointestinal tract, the experiment as shown in Figure 9A was repeated in cells from a distal (colon) gastrointestinal tumor (DLD-I). In these cells, viability was hardly affected by vitamin D3 treatment, and the expression of the abovementioned did not have an ef- feet. Summarizing, vitamin D3 acts specifically on adenocarcinomas of the proximal gastrointestinal tract and does so by acting on the Hh pathway
Legend to the figures Figure 1. Cholesterol synthesis and possible modes of action of Ptchl
(A) Schematic representation of cholesterol synthesis. Boxed are lathosterol and 7-dehydrocholesterol (7-DHC) , cholesterol precur-
sors known to accumulate in lathosterolosis and Smith-Lemli-Opitz (SLOS) patients respectively. Mutations in, or genetic loss of sterol C5-desaturase {Sc5d) cause stacking of lathosterol, whereas dysfunction of 7-dehydrocholesterol reductase {Dchr7) or the addition of its synthetic inhibitor AY-9944 causes accumulation of 7-DHC. The conversion of 7-dehydrocholesterol to vitamin D3 (cholecalciferol) is mediated by UV light. Statins, like pravastatin, inhibit 3-hydroxy-3- methylgluatryl coenzyme A reductase, the enzyme that forms me- valonate . (B) Three possible models for the inhibitory action of the Hh receptor patched (Ptchl) on smoothened (Smo) . 1) A cell autonomous mode of action, in which direct binding of Ptchl inhibits Smo. 2) An intracellular inhibitory action, mediated by direct binding of Ptchl to Smo. 3) The model in which Ptchl pumps an inhibitory small mole- cule that is capable of Smo-repression intercellularly (as well as intracellularly, not shown) .
Figure 2. Confirmation of functionality of constructs and model system used (A) Transfection of Ptchl expression construct in C3H/10T1/2 fibroblasts increased Ptchl expression over basal expression as seen on Western blot. Actin levels remained unaltered, cells were lysed 24h post-transfection.
(B) C3H/10T1/2 cells are sensitive to Hh pathway components as indicated by Gli-reporter activity when pathway components are expressed; Smo increased Hh pathway activity as determined by GIi reporter luciferase assay. Cotransfection of Ptchl suppressed Smo- induced GIi activation. Transfection of Ptchl in the absence of Smo overexpression did not decrease GIi activity below control levels. Shh stimulation (1 μg/ml for 6h, 16h post transfection) , and transfection of a Glil expression construct showed highest reporter activity as expected. Addition of 1 μg/ml Shh-blocking antibody 5El reduced Shh-mediated activation of Gli-reporter activity. The Ptchl- insensitive mutant SmoM2 showed a high basal activity which could not be diminished by co-transfecting Ptchl as expected. Cells were trans- fected and lysed after 24h. Values shown are RLU values corrected for an internal CMV Renilla standard, expressed as percent increase rela-
tive to vector (pcDNA 3.1-) transfection or control stimulation. Depicted is the mean +/- SEM. (n = 4; * = p<0.05; ** = p<0.01).
(C) Shh concentration in medium is below the detection limit (5 ng/ml) of Western blotting. Medium was spiked with decreasing concen- tratioήs of recombinant Shh, and blotted along with a 4x concentrated medium sample obtained from C3H/10T1/2 fibroblasts (incubated for 16h at a volume to surface ratio identical to the Mix-and-Match and medium transfer experiments) .
Figure 3. Ptchl inhibits Smo intercellularly
(A) Detaching and mixing of two populations of fluorescently labelled cells resulted in a homogenous mixture with intimate cell- cell contacts. The procedure is summarized in the left panel. Original magnification 400X. Intercellular capacity of Ptchl to inhibit Smo was assessed by mixing reporter cells overexpressing Smo and a Gli-reporter with Ptchl overexpressing donor cells . The observed decrease in Gli-reporter activity could not be inhibited by the addition of 1 μg/ml 5El Shh-blocking antibody. Exogenously added Shh (1 μg/ml) was capable of abolishing the Ptchl mediated inhibition. Transfecting donor cells with Ptchl siRNA reversed the inhibitory effect (blue bars). MDA-MB-231 breast carcinoma cells (hatched bars) showed a similar response as the C3H/10T1/2 fibroblasts. Donor cells were transfected, washed (16h post transfection), mixed with reporter cells and lysed after 6-8h. Shown are RLU values corrected for an in- ternal CMV Renilla luciferase control, expressed as percent differences relative to control transfected donor cells (vector DNA or scrambled control siRNA) . (n ≥ 4; * = p<0.05; *** = p<0.005). Depicted is the mean +/- SEM.
(B) Fusion of reporter cells with Ptchl overexpressing donor cells showed a decrease in Gli-reporter activity comparable in magnitude to the inhibition observed in mixed cells. Right panel; treating fluorescently labelled cells with PEG1500 4h after mixing resulted in multiple nuclei per cell and composite cell colours, indicating successful cell fusion. Magnification 100OX. Statistics and incubation times as (A)
(C) To assess the specificity of Ptchl acting on Smo, reporter cells expressing the Ptchl-inse'nsitive mutant SmoM2 (as indicated on the y-axis) were mixed with donor cells. The loss of inhibition indi-
cates the specificity of Ptchl acting through Smo in the Mix-and- Match setup. Statistics and incubation times as (A)
(D) Model for the proposed intercellular action of Ptchl inhibition following the results obtained in the Mix-and-Match experi- ments.
Figure 4. Ptchl secretes Smo-inhibitory 3β-hydroxysteroids
(A) Left panel; Schematic representation of the medium transfer experiments, in which medium was incubated on (transfected) donor cells and subsequently transferred to reporter cells. Right panel; Intercellular Smo inhibition by Ptchl is carried by the medium. Medium conditioned by control-, Ptchl- or Ptchl siRNA transfected (C3H/10T1/2, solid bars and MDA-MB-231, hatched bars) cells was transferred to Gli-reporter cells. The same inhibitory action for Ptchl was found as in the Mix-and-Match experiments . Neither 5El blocking antibody nor recombinant Shh addition to the reporter cells could diminish the inhibitory potential of the conditioned media. Medium conditioned on wild type (Ptchl+/+) MEFs showed a pronounced inhibitory effect on reporter cells as compared to Ptchl knockout {Ptchl-/-) MEF conditioned medium. GIi activity in reporter cells transfected with SmoM2 was not inhibited by Ptc +/+ MEF conditioned medium. In the absence of serum, Ptchl transfectant conditioned medium did not contain the inhibitory activity. Incubation times as Figure 3B. Values shown are RLU values corrected for an internal CMV Renilla standard and expressed as percent difference to control transfected donor cells. Depicted is the mean +/- SEM. (n ≥ 4; * = p<0.05; ** = p<0.01 medium Ptchl-transfected compared to medium Ptchl siRNA-transfected) .
(B) Vector, Ptchl, and Ptchl siRNA transfectant conditioned me- dia investigated with FPLC-coupled CHOD-PAP analysis shows loading of
3β-hydroxysteroids on lipoproteins by control cells . Medium conditioned by Ptchl transfected cells shows a strong increase in 3fi- hydroxysteroids, mainly in the LDL fraction. Ptchl siRNA transfection abolishes 3β-hydroxysteroid loading on LDL. The inset shows the lipid standard FCS, containing VLDL, LDL and HDL. Medium was incubated on cells for 16h. Shown are typical profiles.
(C) Quantification of the LDL peaks expressed as mM LDL-C shows the Ptchl induced increase, a reduction by HMG-CoA reductase inhibi-
tor pravastatin treatment and the abolition by Ptchl siRNA transfec- tion of 3β-hydroxysteroid loading on LDL (n = 3; * = p<0.05; ** = p<0.01) .
(D) Pravastatin inhibited Ptchl action on Smo-driven GIi re- porter activity, whereas addition of the cholesterol precursor me- valonate enhanced this inhibition. Cells were transfected and after 16h, pravastatin or mevalonate was added for 6-8h. Depicted is the mean +/- SEM. (n = 4; * = p<0.05; ** = p<0.01).
(E) Hh-induced endocytosis is not inhibited by 1 mM pravastatin (n=24; *** = p<0.005) . Cells were stimulated with 1 μg/ml Shh for Ih, and preincubated with 1 mM pravastatin or control for 6h.
Figure 5. Differentially modulated Ptchl action in Dhcr7-/- and ScSd-/- MEFs (A) Basal GIi reporter activity measured in MEFs either deficient for Sc5d or Dhcr7 shows that the stacking of 7-DHC (in the Dhcr7-/~ MEFs) lowers GIi activity in the mutant cells compared to lathosterol stacking in the Sc5d -/- MEFs. Transfer of the conditioned media to reporter cells (indicated in Figure as "MEFs as me- dium donors") shows that the inhibitory potential of the Dhcr7-/- MEFs is medium-borne. Media were incubated on donor MEFs for 16h and transferred to reporter cells for 6-8h. (n ≥ 4; * = p<0.05; *** = p<0.005)
(B) Medium transfer of Ptchl, Ptchl siRNA or control trans- fected Sc5d-/- and Dhcrl-/- MEFs shows that cells stacking lathosterol but lacking 7-DHC (Sc5d-/- MEFs) are not able to translate different Ptchl levels to an inhibitory action on reporter cells. The Dhcr7-/- cells were able to properly convey an inhibitory signal when transfected with Ptchl or inversely, show a diminished inhibitory po- tential upon Ptchl siRNA transfection. UVB treatment of Ptchl trans- fectant medium (2h) amplified the inhibitory action. Media incubations and statistics as (A) .
Figure 6. Analysis of vitamin D3 as a specific Smo-antagonist (A) Shown are the Gli-reporter inhibitions by 7-DHC, AY-9944, a Dhcr7 inhibitor, vitamin D3 and a combination of the latter two. Also shown is the inhibition that can be conferred by the well-known Smo- antagonist cyclopamine and the previously observed effects for Ptchl
transfectant conditioned medium as well as Ptchl cotransfection (data taken from 4B Figure 2B respectively) . Cells were stimulated with the various compounds for 6h (16h past transfection) , except for the cotransfection which was lysed after 24h. (n ≥ 4) . (B) A range of concentrations of vitamin D3 was tested for inhibition of GIi reporter activity (also m SmoM2 transfected cells), MTT viability assay, and N-termmal repressor form Gli3 levels. Cells were stimulated with vitamin D3 for 6h prior to lysis. For the reporter assays, values were RLLJ values corrected for an internal CMV Renilla standard and expressed as percent difference to control stimulated cells (n = 4) . The MTT assays shown were raw absorption data expressed as percent difference to control stimulated (n = 8), MTT agent was added foi 3h. N-termmal repressor form Gli3 was determined by quantifying ECL signal from Western blot and corrected for α-actin (n = 3) . Depicted is the mean +/- SEM.
(C) Gli-specificity of the inhibitory effect of 10 μM vitamin D3 was assayed by using a panel of luciferase reporter constructs, amongst which a mutant GIi binding site reporter (mGli-LUC) . Cells were stimulated for 6h with vitamin D3 and the luciferase activity was assayed, values were calculated from RLU values corrected for an internal CMV Renilla standard and expressed as percent difference to control stimulated cells (n ≥ 4) . Depicted is the mean +/- SEM.
(D) Different cell types share the inhibitory response to vitamin D3 on GIi activity. C3H/10T1/2 and MDA-MD-231 cells were used earlier m the Mix-and-Match and medium transfer experiments. Ptchl - /- MEFs served as a control for any possible effects of vitamin D3 on Ptchl function. Cells were stimulated for 6h and the luciferase activity was assayed, values were RLU values corrected for an internal CMV Renilla standard and expressed as percent difference to control stimulated cells (n = 4) . Depicted is the mean +/- SEM.
(E) Proposed mechanism of vitamin D3 action. First panel, m the presence of Hh, Ptchl is inactive, and does not inhibit Smo . The Hh pathway is active and GIi activity can be measured in a reporter assay. Second panel; in the absence of Hh, Ptchl is active and util- izes vitamin D3 to inhibit Smo. The pathway is inactive and low GIi activity is measured. Third panel; m the presence of Hh as well as exogenous D3, Smo is inhibited independently of Ptchl and Hh can no longer elicit a GIi response.
(F) Confluent Shh-LIGHT Il stable GIi reporter transfectants were stimulated with 10 uM of vitamin D3 and/or 200 ng/ml Shh overnight in the presence of 0.5% FCS. Luciferase activity was assayed, values are RLU values corrected for an internal CMV Renilla standard and expressed as percent difference to control stimulated cells (0 uM vitamin D3, 0 ng/ml Shh) . In the presence of vitamin D3, Shh is no longer able to induce reporter activity, n = 4, depicted is the mean +/- SEM.
Figure 7. Scatchard analysis of vitamin D3 using Smo-expressing P±chia pastoris
(A) 24h methanol induction (buffered methanol-complex medium; BMMY) of Smo-transforrαed P. pastoris yielded successful Smo expression as shown by Western blot analysis with an antibody directed against Smo.
(B) Scatchard analysis of heterologous competition with cyclo- pamine representing the binding of [3H]-D3 to Smo-expressing (closed circles, MeOH induced) and non-expressing (open circles, non-induced) yeast strains. The solid line indicates the induced high affinity binding, whereas the dotted line indicates non-specific low affinity binding. Scatchard analysis and fitting was performed as described earlier (Pynaert et al . , 1999).
Figure 8. Vitamin D3 reduces cell viability in upper gastroin- testinal tract cancer cell lines.
(A) A concentration range of vitamin D3 was added to proximal digestive tract tumor cell lines for 24h and cell viability was assayed. A strongly decreased cell viability was observed upon vitamin D3 treatment. (B) Comparison of the cytotoxicity of cyclopamine and D3. At 100 μM, vitamin D3 was more potent in inhibiting cell viability than cyclopamine for gastric and oesophageal cancer cell lines tested.
(C) As (B), using pancreatic and hepatic cancer cell lines.
(D) Addition of 10 μM of the Dhcr7 inhibitor AY-9944 greatly increased the toxic effect of vitamin D3 in tumor cell lines. Treatment with AY-9944 alone reduced cell viability in all but the HMO-2 cell line. Viability assay performed in the presence of 10% FCS.
Figure 9. Specificity of cytotoxicity of vitamin D3 to adenocarcinoma of the proximal gastrointestinal tract with abberant Hh signaling
(A) Pancreatic cancer cells (BXPC3, see also Figure 8) were treated with vitamin D3 as shown on Y-axis and cell viability was assessed. To establish the specificity of vitamin D3 acting through the Hh pathway in these cells, transfections with either a mutant Smo receptor (SmoM2) or the downstream transcription factor (Glil) were performed. Overexpression of either of these diminished the cytotox- icity of vitamin D3 (fold reduction indicated in figure next to bars), indicating the specificity of vitamin D3 acting through the Hh pathway.
(B) The colon cancer cell line DLD-I (distal gastrointestinal tumor) did not show sensitivity to treatment with vitamin D3. The small inhibition of cell growth observed could not be diminished by transfection with either SmoM2 or Glil as expected.
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