1. Volume 133, Issue 4, Pages 1272-1281 (October 2007)
The Effect of Statins in Colorectal Cancer Is Mediated Through the Bone Morphogenetic Protein Pathway 
Liudmila L. Kodach, Sylvia A. Bleuming, Maikel P. Peppelenbosch, Daniel W. Hommes, Gijs R. van den Brink, James C.H. Hardwick 
Gastroenterology 
Volume 133, Issue 4, Pages (October 2007)
DOI: /j.gastro
Copyright © 2007 AGA Institute Terms and Conditions

2. Figure 1
The effects of statins on the growth of CRC cell lines. (A) The MTT cell viability assay was performed in 4 colon cancer cell lines treated for 24 hours with various concentrations of lovastatin. Values are expressed as a percentage of controls. Data are from 3 experiments. ± SEM (n = 10). (B) Immunoblots of SW480, HCT116, HT29, and DLD1 colon cancer cell lines for SMAD4 with β-actin as a loading control. (C) HCT116 and HT29 cells were treated for 24 hours with various concentrations of lovastatin, simvastatin or pravastatin, and the MTT assay was performed as in panel A.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

3. Figure 2
Lovastatin activates the BMP pathway in HCT116 cells, but not in SW480 cells. (A) Immunoblots of colon cancer cells treated with various concentrations of lovastatin (μmol) for 24 hours. The expression of proteins was analyzed using the corresponding specific antibody. β-actin served as a loading control. (B) HCT116 cells were treated with lovastatin, lovastatin in combination with noggin (0.5 μg/mL), or lovastatin in combination with mevalonate (100 μmol) for 24 hours, and apoptosis was quantified by flow cytometry. Data are from 3 experiments ± SEM (n = 3). (C) HCT116 cells were treated with lovastatin or lovastatin in combination with noggin (0.5 μg/mL) for 24 hours and then photographed under a microscope. (D) HCT116 cells were treated with lovastatin or lovastatin in combination with noggin (0.5 μg/mL) for 24 hours and subsequently counted. Values are expressed as a percentage of controls. Data are from 3 experiments ± SEM (n = 4). (E and F) SW480 cells were treated with lovastatin or lovastatin in combination with noggin (0.5 μg/mL) for 24 hours. In (E) apoptosis was quantified by flow cytometry. Data are from 3 experiments ± SEM (n = 3). In (F) cells were counted and values are expressed as a percentage of controls. Data are from 3 experiments ± SEM.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

4. Figure 3
HCT116 cells were treated with 5-fluorouracil (A) or aspirin (B) with or without the simultaneous addition of noggin (0.5 μg/mL) for 24 hours and subsequently counted. Values obtained in the absence of compound have been set at 100. Results represent the mean ± SEM (n = 4) of 3 experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

5. Figure 4
The response of HCT116 SMAD4+/+ and HCT116 SMAD4 −/− cell lines to lovastatin treatment. (A) The MTT assay was performed in HCT116 SMAD4+/+ or HCT116 SMAD4−/− cells treated for 24 hours with various concentrations of lovastatin. Values are expressed as a percentage of controls. Data are from 3 experiments ± SEM ***P < .001 (n = 10). (B) HCT116 SMAD4+/+ or HCT116 SMAD4−/− cells were treated with lovastatin for 24 hours, and apoptosis was quantified by flow cytometry. Data are from 3 experiments ± SEM. *P < .05 (n = 3). (C and D) HCT116 SMAD4+/+ or HCT116 SMAD4−/− cells were transiently transfected with BMP2-Luc vector (C) or the BRE-Luc vector (D) and treated with various concentrations of lovastatin for 24 hours. Data were normalized to Renilla luciferase activity. Data are from 3 experiments ± SEM (n = 4). (D) as in C, except cells were transfected with the BRE-Luc vector. (E) HCT116 SMAD4−/− cells were transiently transfected either with SMAD4 or with GFP construct, treated for 24 hours with various concentrations of lovastatin, and the MTT assay was performed as in A. (F) HCT116 SMAD4−/− cells were transiently cotransfected either with SMAD4 and BRE-Luc or with GFP and BRE-Luc constructs, treated for 24 hours with 2 μmol of lovastatin or 100 ng/mL recombinant human BMP2, and transcriptional activity was measured and normalized to Renilla-luciferase activity. Data are from 3 experiments ± SEM (n = 4).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

6. Figure 5
Growth curves of HCT116 and HT29 implants in nude mice. (A and B) Groups of 8 female NMRI nu/nu mice were injected subcutaneously in the flank with either 1 × 106 HCT116 cells (A) 1 × 106 HT29 cells (B) in Matrigel. Mice were fed ad libitum with food containing simvastatin, thereby receiving 50 mg/kg per day, initiated when the tumor volume reached 100 to 200 mm3. Controls received identical food without simvastatin. Tumor volumes were determined by external measurement. Mice were sacrificed when the largest tumors reached 1000 mm3. Results represent the mean ± SEM. *P < .05, **P < .01. (B), as in A, except 5 × 106 HT29 cells were used, and simvastatin treatment was for 12 days before mice had to be sacrificed due to tumor size. (C) Immunoblot analysis of lysates of HCT116 and HT29 xenografts from mice treated with simvastatin and from controls. Fifty micrograms of total protein was loaded per lane. Equal loading was confirmed by assessing β-actin. Simvastatin treatment increases phosphorylated SMAD1, 5, and 8 expression suggesting activation of BMP signaling. (D) Graph shows the relative mean expression of pSMAD1, 5, and 8 in lysates of HCT116 xenografts from mice treated with simvastatin and from controls analyzed by immunoblotting. Expression in control mice was set at 1. Error bars represent the SEM. ***P < .001.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

7. Figure 6
Low concentrations of lovastatin induce BMP2 and reduce cell viability. (A) HCT116 cells were plated in concentration 1000 and 10,000 per well in the 6-well plates and treated for 3 weeks with lovastatin (0.1 μmol). Cell viability was assessed using the MTT assay. Values are expressed as a percentage of controls. Data are from 3 experiments SEM (n = 10). **P < .01. (B) Immunoblots of HCT116 cells treated with lovastatin (0.15 μmol) for 48 and 72 hours. The expression of BMP2 was analyzed using the corresponding specific antibody. β-actin served as a loading control.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions