1. Volume 129, Issue 3, Pages 952-968 (September 2005)
Gastrin Suppresses Growth of CCK2 Receptor Expressing Colon Cancer Cells by Inducing Apoptosis In Vitro and In Vivo 
Susanne Müerköster, Anett Isberner, Alexander Arlt, Maike Witt, Babette Reimann, Ewelina Blaszczuk, Veronika Werbing, Ulrich R. Fölsch, Frank Schmitz, Heiner Schäfer 
Gastroenterology 
Volume 129, Issue 3, Pages (September 2005)
DOI: /j.gastro
Copyright © 2005 American Gastroenterological Association Terms and Conditions

2. Figure 1
Differential CCK2 receptor expression on human colon cancer cell lines. (A) Total RNA samples from 7 human colon cancer cell lines were subjected to RT-PCR analysis using primers specific for the CCK2 receptor. In parallel, RT-PCR was conducted for β-actin, which was used as a control. (B) Radio ligand-binding assay was performed with Colo320wt, Colo320mut, and Lovo cells. Cells were incubated with 125I-labeled sulphated CCK8 in the absence or presence of increasing concentrations of unlabeled sulphated CCK8 (CCK8), G17, G17gly, or the CCK2 receptor antagonist (L ). One representative experiment out of 3 is shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

3. Figure 2
G17 suppresses proliferation of human colon cancer cells expressing the wild-type but not the mutant CCK2 receptor. (A) Colo320wt, Lovo, and Colo320mut cells were seeded onto 6-well plates. After 24 hours, cell numbers were determined by counting (0 hours), and the cells were either left untreated or treated with 1 nmol/L or 10 nmol/L G17 and G17gly, respectively, each in the absence or presence of 500 nmol/L L Cell numbers were determined 24, 48, 72, and 96 hours after stimulation. One representative experiment out of 3 is shown. (B) Colo320wt, Lovo, and Colo320mut cells were either left untreated or were treated with 1 nmol/L G17 for 24 hours. Measurement of propidium iodide (PI) uptake and cell cycle analysis were done by fluorescence flow cytometry. Means ± SD from 3 independent experiments are shown; *statistical significance (P < .05) of the compared values, as indicated by the frames. (C) Representative histograms show PI uptake (FL2/PI) in Colo320wt and Colo320mut cells that were either left untreated or treated with 1 nmol/L G17. Cell cycle phases of 2n- to 4n-gated cells (diploid/tetraploid cells without sub-G1) are indicated in each histogram.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

4. Figure 2
G17 suppresses proliferation of human colon cancer cells expressing the wild-type but not the mutant CCK2 receptor. (A) Colo320wt, Lovo, and Colo320mut cells were seeded onto 6-well plates. After 24 hours, cell numbers were determined by counting (0 hours), and the cells were either left untreated or treated with 1 nmol/L or 10 nmol/L G17 and G17gly, respectively, each in the absence or presence of 500 nmol/L L Cell numbers were determined 24, 48, 72, and 96 hours after stimulation. One representative experiment out of 3 is shown. (B) Colo320wt, Lovo, and Colo320mut cells were either left untreated or were treated with 1 nmol/L G17 for 24 hours. Measurement of propidium iodide (PI) uptake and cell cycle analysis were done by fluorescence flow cytometry. Means ± SD from 3 independent experiments are shown; *statistical significance (P < .05) of the compared values, as indicated by the frames. (C) Representative histograms show PI uptake (FL2/PI) in Colo320wt and Colo320mut cells that were either left untreated or treated with 1 nmol/L G17. Cell cycle phases of 2n- to 4n-gated cells (diploid/tetraploid cells without sub-G1) are indicated in each histogram.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

5. Figure 3
G17 induces apoptosis in human colon cancer cells expressing the wild-type but not the mutant CCK2 receptor. (A) Colo320wt, Lovo, and Colo320mut cells were either left untreated or treated with 1 nmol/L or 10 nmol/L G17 either in the absence or presence of 500 nmol/L L or with G17gly. After 24 hours of treatment, apoptosis was determined by staining with annexinV/PI followed by fluorescence flow cytometry. Data are presented as percentage apoptotic cells over basal. Means ± SD from 3 independent experiments are shown (top). Representative images from FACS analysis (bottom) show staining intensity for AnnexinV (FL1) and PI (FL2) in Colo320wt and Colo320mut cells. (B) Colo320wt, Lovo, and Colo320mut cells were either left untreated or treated with 10 nmol/L G17 for 24 hours and analyzed for changes in ΔΨm by JC1 staining expressed as changes in the FL2/FL1 ratio. (C) Cells were treated as above and analyzed for caspase-3/7 activity, which is expressed as n-fold induction of caspase-3/7 over basal. Means ± SD from 3 independent experiments are shown. (D) Colo320wt and Colo320mut cells were either left untreated or treated with 10 nmol/L G17 for 48 hours. Representative pictures of TUNEL staining of untreated and G17-treated Colo320wt and Colo320mut cells, respectively, are shown. Magnification 250×; *statistical significance (P < .05) of the compared values, as indicated by the frames.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

6. Figure 3
G17 induces apoptosis in human colon cancer cells expressing the wild-type but not the mutant CCK2 receptor. (A) Colo320wt, Lovo, and Colo320mut cells were either left untreated or treated with 1 nmol/L or 10 nmol/L G17 either in the absence or presence of 500 nmol/L L or with G17gly. After 24 hours of treatment, apoptosis was determined by staining with annexinV/PI followed by fluorescence flow cytometry. Data are presented as percentage apoptotic cells over basal. Means ± SD from 3 independent experiments are shown (top). Representative images from FACS analysis (bottom) show staining intensity for AnnexinV (FL1) and PI (FL2) in Colo320wt and Colo320mut cells. (B) Colo320wt, Lovo, and Colo320mut cells were either left untreated or treated with 10 nmol/L G17 for 24 hours and analyzed for changes in ΔΨm by JC1 staining expressed as changes in the FL2/FL1 ratio. (C) Cells were treated as above and analyzed for caspase-3/7 activity, which is expressed as n-fold induction of caspase-3/7 over basal. Means ± SD from 3 independent experiments are shown. (D) Colo320wt and Colo320mut cells were either left untreated or treated with 10 nmol/L G17 for 48 hours. Representative pictures of TUNEL staining of untreated and G17-treated Colo320wt and Colo320mut cells, respectively, are shown. Magnification 250×; *statistical significance (P < .05) of the compared values, as indicated by the frames.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

7. Figure 4
Colo320wt and Colo320mut tumors respond differently upon hypergastrinemia. (A) Colo320wt and Colo320mut tumor-bearing animals were randomized each into 2 groups (n = 5) and subjected to treatment with 0.9% NaCl or omeprazole. Animals were killed for further analysis at day 34 after tumor inoculation, and tumor sizes were determined. (B) For detection of apoptotic cells, the TUNEL assay was performed with frozen tumor sections from each group. Results were evaluated by counting positively stained nuclei per microscopic field (magnification ×250). Means ± SD of 2 independent experiments are shown; *statistical significance (P < .05) of the compared values, as indicated by the frames. (C) Representative pictures of TUNEL staining in frozen tissue sections of NaCl and omeprazole-treated Colo320wt and Colo320mut tumors, respectively, are shown. Arrows indicate regions with abundant apoptotic cells.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

8. Figure 5
Proapoptotic effect of gastrin is mediated via the wild-type CCK2 receptor in vivo. (A) Animals with subcutanously grown Colo320wt tumors were randomized into 4 groups (n = 5) and were left untreated (NaCl/NaCl) or treated with omeprazole (omeprazole/NaCl), with L (NaCl/L ), or with a combination of both reagents (omeprazole/L ). Animals were killed for further analysis at day 34 after tumor inoculation, and tumor sizes were determined. (B) For detection of apoptotic cells, the TUNEL assay was performed with frozen tumor sections from each group. Results were evaluated by counting positively stained nuclei per microscopic field (magnification ×250). Means ± SD of 2 independent experiments are shown; *statistical significance (P < .05) of the compared values, as indicated by the frames. (C) Representative images of TUNEL staining of frozen tissue sections from untreated (NaCl/NaCl) Colo320wt tumors or tumors treated with omeprazole (omeprazole/NaCl), L (NaCl/L ), or a combination of both (omeprazole/L ) are shown. Arrows indicate regions with abundant apoptotic cells.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

9. Figure 6
Activation of the MAPK/ERK pathway by G17 in Colo320wt cells. (A) Colo320wt and Colo320mut cells were either treated with G17 at the indicated doses for 5 minutes or not. Cellular lysates were submitted to Western blotting using a phospho-Erk1/Erk2 antibody or an α-tubulin antibody as loading control. (B) Colo320wt and Colo320mut cells were either treated with 10 nmol/L G17 for the indicated periods or not. Total RNA was submitted to RT-PCR analysis using specific primers for cFos or β-actin as control. (C) Colo320wt and Colo320mut cells were either treated with 0.1 nmol/L or 10 nmol/L G17 for 1 hour or not. Nuclear extracts from these cells were submitted to gel-shift assay for the detection of AP-1-binding activity. All results are representative from 1 of 3 independent experiments. u.b., unspecific band. (D) Colo320wt and Colo320mut cells were transfected with an AP-1 luciferase reporter gene vector (p5xTRE-Luc) or an empty vector (pGL3) together with the pRLTK vector. Transfectants were either treated with 0.1 and 10 nmol/L G17 for 6 hours or not, and cell lysates were submitted to a dual luciferase assay. Firefly-luciferase expression was normalized to the amount of renilla-luciferase (pRLTK), and data represent the specific AP-1-driven luciferase expression (from the normalized p5xTRELuc/pGL3Luc ratios). Means ± SD of 3 independent experiments performed in duplicates are shown; *statistical significance (P < .05) of the compared values, as indicated by the frames.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

10. Figure 7
Inhibition of the IκBα/NF-κB pathway by G17 in Colo320wt cells. (A) Colo320wt and Colo320mut cells were either left untreated or treated with 10 nmol/L G17 for 1 hour, with 30 ng/mL TNF-α for 30 minutes, or with a combination of both. Nuclear extracts from these cells were submitted to gel-shift assay for the detection of NF-κB binding activity. u.b., unspecific band. (B) Colo320wt and Colo320mut cells were transfected with a NF-κB luciferase reporter gene vector (pκB-Luc) or an empty vector (pGL3) together with the pRLTK vector. Transfectants were left untreated or treated for 6 hours with 0.1 and 10 nmol/L G17 or with 30 ng/mL TNF-α, either alone or after preincubation with 10 nmol/L G17 for 30 minutes. Cell lysates were submitted to a dual luciferase assay. Firefly luciferase expression was normalized to the amount of renilla-luciferase (pRLTK), and data represent the specific NF-κB-driven luciferase expression (from the normalized pκBLuc/pGL3Luc ratios). Means ± SD of 3 independent experiments performed in duplicates are shown. (C) Colo320wt and Colo320mut cells were left untreated or treated with 30 ng/mL TNF-α for the indicated periods, either alone or after preincubation with 10 nmol/L G17 for 30 min. In addition, cells were treated with the 26S proteasome inhibitor Mg132 (0.5 μmol/L) 30 minutes prior to TNF-α administration (center and lower panels). Cellular lysates were submitted to Western blotting using IκBα and phospho-IκBα antibodies or an α-tubulin antibody as loading control. (D) Colo320wt and Colo320mut cells were left untreated or treated with 10 nmol/L G17, 30 ng/mL TNF-α, or 0.5 mmol/L sulfasalazine, either alone or in the indicated combinations for 24 hours. Apoptosis was determined by staining with annexin V/PI and by fluorescence flow cytometry. Means ± SD from 3 independent experiments are shown; *statistical significance (P < .05) of the compared values, as indicated by the frames.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

11. Figure 8
Inhibition of ERK activation alters the NF-κB-inhibiting and the apoptosis-inducing effect of G17. (A) Colo320wt cells were left untreated or treated with 10 nmol/L G17 for 1 hour, 30 ng/mL TNF-α for 30 minutes, or with 25 μmol/L PD98059 for 1 hour, either alone or in the combination as indicated. Nuclear extracts from these cells were submitted to gel-shift assay for the detection of NF-κB-binding activity. (B) Colo320wt cells were left untreated or treated with 10 nmol/L G17, 30 ng/mL TNF-α, or with 25 μmol/L PD98059, either alone or in the indicated combinations for 24 hours. Apoptosis was determined by staining with annexin V/PI and by fluorescence flow cytometry. Data are presented as percentage apoptotic cells over basal. Means ± SD from 3 independent experiments are shown; *statistical significance (P < .05) of the compared values, as indicated by the frames.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions