Gastrin 1–6 promotes growth of colon cancer cells through non-CCK receptors

Copps, Jeffrey; Ahmed, Shawn; Murphy, Richard F.; Lovas, Sándor

doi:10.1016/j.peptides.2006.10.008

Cited by 6 publications

(13 citation statements)

References 12 publications

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“…In our previous study, we showed that G17(1–12) binds to two sites on DLD-1 cells with similar affinities to that of G17-Gly[11]. We further truncated G17 to produce G17(1–6)-NH 2 to find that it binds to DLD-1 cells at a single site with micromolar affinity and it also can stimulate cell proliferation.…”

Section: Introductionmentioning

confidence: 99%

Bioactivity of analogs of the N-terminal region of gastrin-17

et al. 2009

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Gastrin-17-Gly (G17-Gly) has been shown to bind to non-CCK nanomolar and micromolar affinity sites on DLD-1 and HT-29 human colonic carcinoma cells and to stimulate cellular proliferation. However, in previous studies, we showed that C-terminal truncation of the gastrin-17 (G17) to the G17 analog G17(1–12) and then to G17(1–6)-NH2 did not remove the ability to bind to DLD-1 cells or to activate proliferation. This implies that residues and/or structural motifs required for bioactivity at these receptors rest in the N-terminal region of G17. In this work, radioligand binding studies conducted with further C-terminally truncated analogs revealed that sequences as short as G17(1–4) still bind to a single receptor with micromolar affinity. Additionally, cell proliferation assays showed that G17(1–12) stimulates proliferation of DLD-1 cells, as of HT-29 cells, but the sequences shorter than G17(1–6)-NH2, including nonamidated G17(1–6), were incapable of stimulating proliferation. These observations indicate that the tetrapeptide pGlu-Gly-Pro-Trp is the minimum N-terminal sequence for binding to the probable growth-promoting site on DLD-1 cells. Since analogs shorter than G17(1–6) are able to bind the receptor, these peptides may be of use for developing selective antagonists.

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Section: Introductionmentioning

confidence: 99%

Bioactivity of analogs of the N-terminal region of gastrin-17

et al. 2009

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“…However, this does not explain the inability of the G17(1-6) analog to activate the receptor. Results from studies both of biological activity and structure of the two peptides are similar aside from the results of proliferation assays and ECD studies [13, 15]. The slightly greater tendency of G17(1-6)-NH 2 to form helix/turn structure (as seen in the ECD studies) may be due to stabilization of the peptide structure by the C-terminal amide, and this structural difference, though not detectable by qualitative review of the VCD/IR spectra or REMD simulations, may account for the difference in activity.…”

Section: Discussionmentioning

confidence: 95%

“…The CDSSTR results, however, show that G17(1-6)-NH 2 has a greater percentage of helix/turn structure in these solvents than does G17(1-6) (Table 2). In lieu of other structural and binding differences, if indeed these conformational features represent the bioactive conformation of G17(1-6)-NH 2 , then they may be why G17(1-6)-NH 2 stimulates proliferation while G17(1-6) does not [13, 15]. …”

Section: Discussionmentioning

confidence: 99%

“…It was also demonstrated that the N-terminal region of G17, G17(1-12), was able to stimulate the proliferation of HT-29 cells, showing that the C-terminal tetrapeptide (Trp-Met-Asp-Phe-NH 2 ) of G17 which is essential for binding and activation of the CCK2 receptor is not necessary for binding and activation of the putative G17-Gly growth promoting receptor [3]. Furthermore, we have shown [13] that 1, G17(1-12) binds to high and low affinity receptors on DLD-1 cells as does [Leu 15 ]G17-Gly and promotes the proliferation of such cells; 2, further excision of the sequence Glu 7 -Tyr 12 still gives a peptide, G17(1-6)-NH 2 , which binds, albeit with low affinity, and activates the putative growth promoting receptor on DLD-1 cells; 3, shorter N-terminal analogs with and without C-terminal amidation, including G17(1-6), bind with decreasing affinity than that of G17(1-6)-NH 2 [15]. Sequences shorter than G17(1-4) fail to stimulate proliferation of DLD-1 cells.…”

Section: Introductionmentioning

confidence: 92%

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The structure of bioactive analogs of the N-terminal region of gastrin-17

2009

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Gastrin-17 (G17) processing intermediates bind to non-CCK receptors which mediate growth of the colonic mucosa but also the formation and development of colonic cancers. In previous studies, we removed the C-terminal region of G17 to form G17(1-12) and considerably shorter C-terminally amidated and non-amidated analogs. Peptides as short as G17(1-4) continued to bind to a single site on DLD-1 human colonic carcinoma cells, while only the G17(1-6)-NH 2 and G17(1-12) peptides retained the ability to activate the receptor and stimulate cell proliferation in vitro. In this report, we studied the structure of these analogs, using a combination of ECD and VCD spectroscopy and replica exchange Molecular Dynamics (REMD) simulations in water, TFE, and membrane-mimicking environments, in order to determine preferred conformations that may have importance in promoting the biological activities. Mostly random meander structures, punctuated by a β-turn at residues 1-4, were found in most peptides by REMD simulations. G17(1-3)-NH 2 , which cannot form a β-turn, failed to bind the non-CCK receptor, suggesting the importance of this feature for binding. Additionally, the β-turn appeared more frequently longer sequences, perhaps explaining the higher affinity of the non-CCK receptor for these peptides seen previously. Finally, C-terminally amidated peptides generally showed greater formation of turn structure than their non-amidated counterparts as shown ECD spectra, suggesting the importance of peptide length in stabilizing turn structure in N-terminal sequences, and perhaps explaining the ability of G17(1-6)-NH 2 to activate the non-CCK receptor where as the non-amidated G17(1-6) and shorter peptides do not.

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“…In colonic mucosa, G17 is supposed to trigger proliferation as well, thus for a long time G17 was under suspect to promote colorectal tumorigenesis. However, this view has been substantially changed due to a line of experimental evidence: (1) progastrin or Gly-gastrin rather than G17 exerts a strong proliferative effect on various experimental settings in a CCK2 receptor-independent fashion (Siddheshwar et al, 2001;Ahmed et al, 2004;Cobb et al, 2004;Singh et al, 2007), (2) little evidence exists for simultaneous expression of mature G17 and the CCK2 receptor in colon cancer cells (Clerc et al, 1997;Copps et al, 2006) and (3) a rather growth inhibitory and proapoptotic effect of G17 on colon cancer cells has been reported in vitro and in vivo Yu et al, 2005).…”

Section: Discussionmentioning

confidence: 99%

The apoptosis-inducing effect of gastrin on colorectal cancer cells relates to an increased IEX-1 expression mediating NF-κB inhibition

et al. 2007

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Addressing the puzzling role of amidated gastrin 17 (G17) and the gastrin/CCKB/CCK2 receptor in colorectal carcinogenesis, we analysed potential candidate genes involved in G17-dependent NF-jB inhibition and apoptosis. The colorectal carcinoma cell line Colo320 overexpressing the wild-type CCK2 receptor (Colo320wt) underwent G17-induced apoptosis along with suppressed NF-jB activation and decreased expression of the antiapoptotic NF-jB target genes cIAP1 and cIAP2, whereas G17 was without effect on Colo320 cells expressing a CCK2 receptor bearing a loss of function mutation (Colo320mut). Gene microarray analysis revealed an elevated expression of the stress response gene IEX-1 in G17-treated Colo320wt but not Colo320mut cells. Quantitative real-time PCR and conventional RT-PCR confirmed this G17-dependent increase of IEX-1 expression in Colo320wt cells. If these cells were subjected to IEX-1 knockdown by small interfering RNA transfection, the apoptosis-inducing effect of G17 was abolished. Moreover, tumor necrosis factor alpha (TNFa)-or 5-FUinduced apoptosis that is greatly enhanced by G17 treatment in Colo320wt cells was prevented if IEX-1 expression was repressed. Under these conditions of blocked IEX-1 expression, the NF-jB activity remained unaffected by G17, in particular in Colo320wt cells co-treated with TNFa and also the suppressive effect of G17 on cIAP1 and cIAP2 expression was not observed anymore if IEX-1 expression was blocked. Conversely, IEX-1 overexpression in Colo320mut cells caused an increase of basal and TNFa-or 5-FU-induced apoptosis, an effect not further triggered by G17 treatment. Using a xenograft tumor model in severe combined immune deficiency mice, we could show that experimental systemic hypergastrinemia induced by the administration of omeprazole led to enhanced apoptosis as well as to a marked increase of IEX-1 expression in Colo320wt tumors, but not in Colo320mut tumors. These observations indicate that the proapoptotic effect of G17 on human colon cancer cells expressing the wild-type CCK2 receptor is mediated by IEX-1, which modulates NF-jB-dependent antiapoptotic protection and thereby exerts tumor-suppressive potential.

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Gastrin 1–6 promotes growth of colon cancer cells through non-CCK receptors

Cited by 6 publications

References 12 publications

Bioactivity of analogs of the N-terminal region of gastrin-17

Bioactivity of analogs of the N-terminal region of gastrin-17

The structure of bioactive analogs of the N-terminal region of gastrin-17

The apoptosis-inducing effect of gastrin on colorectal cancer cells relates to an increased IEX-1 expression mediating NF-κB inhibition

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