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Published in final edited form as: Regul Pept. 2009 Jul 23;158(0):26–31. doi: 10.1016/j.regpep.2009.07.010

BOMBESIN ENHANCES TGF-β GROWTH INHIBITORY EFFECT THROUGH APOPTOSIS INDUCTION IN INTESTINAL EPITHELIAL CELLS

Xianghua Liu a, Junmei Zhao b, Fazhi Li b, Yan-shi Guo b, Mark R Hellmich b, Courtney M Townsend Jr b, Yanna Cao a, Tien C Ko a
PMCID: PMC3894738  NIHMSID: NIHMS134305  PMID: 19631696

Abstract

Mammalian intestinal epithelium undergoes continuous cell turn over, with cell proliferation in the crypts and apoptosis in the villus. Both transforming growth factor (TGF)-β and gastrin-releasing peptide (GRP) are involved in the regulation of intestinal epithelial cells for division, differentiation, adhesion, migration and death. Previously, we have shown that TGF-β and bombesin (BBS) synergistically induce cyclooxygenase-2 (COX-2) expression and subsequent prostaglandin E2 (PGE2) production through p38MAPK in rat intestinal epithelial cell line stably transfected with GRPR (RIE/GRPR), suggesting the interaction between TGF-β signaling pathway and GRP-R. The current study examined the biological responses of RIE/GRPR cells to TGF-β and BBS. Treatment with TGF-β1 (40 pM) and BBS (100 nM) together synergistically inhibited RIE/GRPR growth and induced apoptosis. Pretreatment with SB203580 (10 µM), a specific inhibitor of p38MAPK, partially blocked the synergistic effect of TGF-β and BBS on apoptosis. In conclusion, BBS enhanced TGF-β growth inhibitory effect through apoptosis induction, which is at least partially mediated by p38MAPK.

Keywords: p38MAPK, Gastrin-releasing peptide receptor, Cell cycle

INTRODUCTION

The intestinal epithelium is one of the most rapidly proliferating tissues in the body. It forms a continuous two-dimensional sheet where new cells are added in the crypts and subsequently removed by apoptosis upon reaching the villus tips three to eight days later [1, 2]. Renewal of the intestinal epithelium involves a complex, dynamic cellular process that is directionally oriented along the crypt-villus axis. Once intestinal epithelial cells migrate to the villus, they undergo cell cycle arrest, differentiation, and eventually apoptosis [3, 4]. In normal tissue, renewal of the intestinal epithelium is controlled by signaling pathways that regulate cell division, survival, motility and differentiation. Alteration of these signaling pathways leads to gastrointestinal diseases [5].

Transforming growth factor (TGF)-β is involved in the regulation of epithelial cells on division, differentiation, adhesion, migration and death [6–8]. TGF-β inhibits proliferation of crypt cells in the small intestine and colon in vivo [9], and inhibits proliferation of intestinal epithelial cells in vitro [10–12]. TGF-β stimulates the migration of intestinal epithelial cells [13, 14] and induces their differentiation [15]. TGF-β also plays an important role in mediating apoptosis in intestinal epithelial cells [16, 17]. In normal epithelial cells and early tumors, TGF-β acts as a tumor suppressor; however, during tumor progression, TGF-β assumes the role as a tumor promoter. Most colon cancers are resistant to the antiproliferative and apoptotic effects of TGF-β, due to loss or mutations of components in the TGF-β signaling pathway, notably TGF-β type I and type II receptors [18] and Smad4 [19, 20].

Bombesin (BBS), a tetradecapeptide, was originally isolated from the skin of the frog Bombina bombina [21]. Later on the mammalian homologue of BBS was identified and named gastrin-releasing peptide (GRP) [22]. In normal tissue, GRP is present in the brain and central nervous system, and the nerve fibres throughout the gastrointestinal tract and pancreas, thymus, prostate, and urethra. The expression of GRPR has also been shown in various fetal or adult tissues, such as the cells lining the gastric antrum [23]. The GRP-like peptides play many physiological roles in addition to the stimulation of gastric acid secretion. They stimulate the release of a variety of hormones including gastrin, somatostatin, CCK, and exocrine secretion from the pancreas, promote smooth muscle contraction [23] and wound repair [24]. GRP/GRPR has been found to mediate the itch sensation in the spinal cord [25] and contribute to intestinal villus growth [26]. Bombesin is a potent gastroprotective agent to prevent gastric injury via the release of endogenous gastrin [27]. It also reduces endotoxemia, intestinal oxidative stress, and apoptosis in experimental obstructive jaundice [28].

We have recently reported that TGF-β and BBS synergistically induced cyclooxygenase-2 (COX-2) expression and subsequently prostaglandin E2 (PGE2) secretion in RIE/GRPR cell line expressing GRPR [29]. These effects are mediated in part by p38MAPK and suggest that the TGF-β signaling pathway interacts with that of the GRPR. However, whether this interaction leads to functional consequences is unknown. To address these questions we used RIE/GRPR cells in this study as a model to examine the effects of TGF-β and BBS on cell growth, apoptosis, and cell cycle progression, and the role of p38MAPK in mediating these effects.

MATERIALS AND METHODS

Reagents

BBS (Bachem Inc, Torrance, CA) was dissolved in ethyl alcohol and diluted in PBS. TGF-β1 (R&D systems Inc, Minneapolis, MN) was diluted in vehicle (0.1% BSA, 4 mM HCl). SB203580 (Calbiochem, La Jolla, CA) was diluted in dimethyl sulfoxide.

Cell lines

Rat intestinal epithelial cell lines stably expressing GRPR (RIE/GRPR) [29] were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Mediatech Inc., Herndon, VA) supplemented with 5% dialyzed fetal bovine serum (dFBS, Invitrogen Co., Carlsbad, CA) and G418 (Mediatech Inc., Herndon, VA) at 400 µg/ml.

Growth study

RIE/GRPR cells were seeded at 5 × 104 cells/well in 12-well plates and cultured for 24 h. On next day, cells were treated with TGF-β (40 pM), BBS (100 nM) alone or together for up to 7 days. Cells were trypsinized and counted daily as described before [30].

Apoptosis assay

DNA fragmentation was quantified by cell death detection ELISA assay (Roche Molecular Biochemicals, Indianapolis, CA) according to manufacturer’s instruction. The number of cells in each condition was adjusted as 5 × 105 cells/ml in lysis buffer provided by the manufacturer. The absorbance (A405–490 nm), an indication of DNA fragmentation, of each sample was measured using a 96-well plate reader (Molecular Devices Co., Sunnyvale, CA) [17]. Apoptotic cells were stained with Annexin V-FITC and propidium iodide using BD ApoAlert™ Annexin V-FITC Apoptosis kit (BD Bioscience, CA). Cells were analyzed by flow cytometry and cells stained only with Annexin V were identified as apoptotic cells as previously described [30].

Cell cycle analysis

The cells were seeded at 1.5 × 105 in 100-mm dishes and incubated at 37°C for 3 days, followed by serum starvation for 2 days. The cells were replated with the dilution of 1/5 and incubated at 37°C for 90 min, and then treated with BBS or TGF-β (40 pM) up to 24 h. The cells were pulsed with 5-bromo-2’ deoxyuridine (BrdU) for the last 45 min and harvested for flow cytometric analysis using BrdU flow kit (BD Biosciences, San Jose, CA) according to the manufacturer’s instruction [30].

Western blotting analysis

Cultured cells were lysed in 1X cell lysis buffer (Cell Signaling Technology, Inc., MA). Protein concentrations of cell lysates were quantified using a protein assay dye (Bio-Rad Laboratories, CA). Western blotting was performed as previously described [17]. Antibodies of p-p38MAPK (phospho-p38MAPK), p38MAPKα, and α-tubulin were from Cell Signaling Technology, Inc., Upstate Biotechnology, and Santa Cruz Biotechnology, Inc. respectively. Horseradish peroxidase (HRP) conjugated goat anti-rabbit and anti-mouse antibodies were from Bio-Rad Laboratories.

Kinase activity assay

RIE/GRPR cells were pretreated with or without SB203580 (10 µM) for 30 min followed by treatment with or without TGF-β (40 pM) for 60 min. Cell lysates were analyzed for p38MAPK activity using a recombinant MAPK-activated protein (MAPKAP) kinase-2 as a p38MAPK substrate (Upstate Biotechnology, Lake Placid, NY) [31].

Statistical Analysis

Data were expressed as means ± SEM. Differences between groups were analyzed by ANOVA with Tukey-Kramer multiple comparisons test, and p< 0.05 is considered significant. Jin’s formula, Q=Ea+b/(Ea+Eb-Ea×Eb), was used to evaluate the combined effects of the drugs on cell growth inhibition [32, 33]. Q is the combination index; Ea+b represents the cell growth inhibition rate of the combined drugs; Ea and Eb represent the cell growth inhibition rate of individual drug. Q value <0.85, from 0.85 to 1.15, and >1.15 represents antagonistic, additive, and synergistic effect respectively.

RESULTS

TGF-β and BBS synergistically inhibit growth of RIE/GRPR cells

A seven-day growth study was conducted to determine whether TGF-β and BBS cooperate to regulate cell growth in RIE/GRPR. Treatment with either TGF-β or BBS alone significantly inhibited cell growth. The combined treatment with both peptides resulted in greater growth suppression than treatment with either peptide alone from day 1 to day 5 (Fig. 1). Most notably, the combined treatment of BBS and TGF-β decreased cell number by 72.6% on day 3, which was significantly lower than either BBS (44.9%) or TGF-β (32.8%) treatment alone. According to Jin’s formula [32], the Q values were above 1.15 from day 3 to day 6, suggesting TGF-β and BBS synergistically inhibit RIE/GRPR cell growth.

Figure 1. TGF-β and BBS inhibited cell growth in RIE/GRPR cells.

Figure 1

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The cells were seeded in 12-well plates for 24 h, then treated with TGF-β1 (40 pM), BBS (100 nM), or TGF-β1 (40 pM) and BBS (100 nM) together. Cell numbers were obtained from triplicate wells and expressed as mean±SEM. *p < 0.05 compared with the group of vehicle control. † p<0.05 compared with either TGF-β1 or BBS alone.

TGF-β and BBS synergistically induce apoptosis in RIE/GRPR cells

Previously, we have shown that TGF-β inhibits growth of intestinal epithelial cells by cell cycle arrest [11, 34] and apoptosis induction [17, 35]. We determined whether the synergistic inhibition of cell growth by TGF-β and BBS is due to increased apoptosis or cell cycle arrest. RIE/GRPR cells were treated with TGF-β and BBS and apoptosis was quantified using a DNA fragmentation ELISA. TGF-β treatment alone induced DNA fragmentation (2.6-fold) compared to the control group (Fig. 2A). However, BBS treatment alone did not induce DNA fragmentation. Treatment with BBS and TGF-β together increased DNA fragmentation by about 6.0-fold compared to the control group (Fig. 2A). Using Annexin V-FITC staining, we found that either TGF-β or BBS alone induced apoptosis; the combined treatment of TGF-β and BBS induced greater apoptosis than either alone (Fig. 2B). Apparently Annexin V-FITC staining was more sensitive to detect an earlier event of apoptosis induced by BBS alone than DNA fragmentation ELISA. Nevertheless, both assays demonstrated the synergistic induction of apoptosis by the combined treatment of TGF-β and BBS.

Figure 2. TGF-β and BBS synergistically induced apoptosis in RIE/GRPR cells.

Figure 2

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A. The cells were seeded in 12-well plates for 24 h, then treated with TGF-β1 (40 pM), BBS (100 nM), or TGF-β1 (40 pM) and BBS (100 nM) together for 24 h. A. DNA fragmentation was quantified by a cell death detection assay. B. Cells were harvested and stained with Annexin V-FITC and propidium iodide. Apoptotic cells with Annexin V-FITC only were expressed as mean±SEM. *p < 0.05 compared with the group of vehicle control, † p < 0.05 compared with the group with treatment of TGF- β alone.

Cell cycle progression was determined in RIE/GRPR cells by growing cells to confluence followed by 48 h serum starvation to induce mitotic quiescence. Cells were then released into the cell cycle by replating in serum-containing media and analyzed for S phase and G0/G1 phase by BrdU incorporation (Fig. 3). The percentages of G0/G1 phase for the control, TGF-β alone, BBS alone, and combination of TGF-β and BBS were 46.4%, 64.0%, 66.9%, and 74.6% respectively (Fig. 3). The percentages of S phase for the control, TGF-β alone, BBS alone, and combination of TGF-β and BBS were 45.0%, 27.7%, 16.5%, and 9.2% respectively (Fig. 3). According to Jin’s formula [32], the Q value is 1.03, suggesting that the cell cycle arrest induced by combined treatment of TGF-β and BBS was an additive effect.

Figure 3. TGF-β and BBS induced cell cycle arrest in RIE/GRPR cells.

Figure 3

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The cells were seeded for 24 h, then starved for 48 h. The cells were treated with TGF-β1 (40 pM), BBS (100 nM), or TGF-β1 (40 pM) and BBS (100 nM) together for 24 h. The cells were pulsed with BrdU during the last 45 min and harvested for flow cytometric analysis. The cells in G0/G1 phase and S phase from duplicate samples were expressed as mean±SEM, *p < 0.05 compared with the group of vehicle control.

It is well known that the cell count reflects the balance between cell proliferation and cell death [36]. In Fig. 2, we demonstrated that the apoptotic changes induced by TGF-β and BBS were from 24 h time point, which accounted for the decreased cell number in the combined treatment groups. The apoptotic changes continued throughout the time course until day 7 when the cells were confluent. Therefore, the most significant changes in cell number observed from days 2 to 5 are a reflection of the cumulative effects of cell lost due to apoptosis. Taken together, our results of apoptosis and cell cycle arrest suggest that TGF-β and BBS cooperatively induced apoptosis in intestinal epithelial cells, resulting in decreased cell number.

p38MAPK partially mediates BBS- and TGF-β-induced apoptosis

Our previous study demonstrated that p38MAPK is involved in the cross-talk of BBS- and TGF-β-induced COX-2 expression in RIE/GRPR cells [29]. To ascertain whether p38MAPK signaling pathway mediates the regulation of cellular function by BBS and TGF-β, we used SB203580, a specific p38MAPK inhibitor to study if p38MAPK is required in apoptosis induction by TGF-β and BBS in RIE/GRPR cells. We found that TGF-β induced phophosphorylation of p38MAPK, whereas pretreatment of SB203580 blocked TGF-β-induced phosphorylation of p38MAPK by western blotting (Fig. 4A). Correspondingly, using a recombinant MAPKAP kinase-2 as a p38MAPK substrate, we measured p38MAPK enzyme activity in vitro. In RIE/GRPR cells, treatment with TGF-β stimulated p38MAPK activity by 1.4 fold. Pretreatment with SB203580 blocked TGF-β-induced p38MAPK activity and decreased the basal level of p38MAPK activity as well (Fig. 4B). Furthermore, pretreatment with SB203580 decreased apoptosis induction by 42.4% in the combination of TGF-β and BBS groups (Fig. 5), suggesting that BBS enhanced TGF-β-induced apoptosis partially through p38MAPK.

Figure 4. SB203580 blocked TGF-β-induced p38MAPK activity.

Figure 4

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RIE/GRPR cells were pretreated with or without SB203580 (10 µM) for 30 min followed by treatment with or without TGF-β (40 pM) for 60 min. A. Whole cell lysates were extracted and subjected to Western blot. p-p38MAPK (phospho-p38MAPK) and p38MAPKα proteins were measured using the specific antibodies, α-tubulin as a loading control. B. Cell lysates were assayed for p38MAPK activity using a recombinant MAPK-activated protein (MAPKAP) kinase-2 as a p38MAPK substrate. Results from duplicate wells were expressed as mean±SEM. *p < 0.05 compared with the group of vehicle control.

Figure 5. SB203580 attenuated TGF-β-induced apoptosis.

Figure 5

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The cells were seeded in 12-well plates for 24 h, then treated for 24 h with TGF-β1 (40 pM), BBS (100 nM), or TGF-β (40 pM) and BBS (100 nM) together, with or without pretreatment of SB203580 (10 µM) for 30 min. DNA fragmentation was quantified by a cell death detection assay. Results from triplicate wells are expressed as mean±SEM. *p < 0.05 compared with the group of vehicle control, † p < 0.05 compared with TGF-β alone, # p < 0.05 compared with the group with both TGF-β and BBS and without SB203580 pretreatment.

DISCUSSION

Growth factors and gastric peptides interact to regulate gut epithelial cell growth, differentiation, and death to maintain gut homeostasis. To investigate the biological responses to TGF-β and BBS in rat intestinal epithelial cells stably-transfected to overexpress GRP receptor (RIE/GRPR), we demonstrated that TGF-β and BBS work synergistically to inhibit RIE/GRPR cell growth and induce apoptosis, and their effects on apoptosis were partially blocked by a p38MAPK inhibitor.

TGF-β controls cellular processes by signaling through cell surface receptor complexes composed of TGF-β type I and type II receptors. Both receptors are transmembrane serine/threonine kinases. TGF-β binds to type II receptor, which subsequently activates the type I receptor by phosphorylation. Once activated, type I receptor phosphorylates Smad2 and Smad3, allowing them bind to Smad4 to form heteromeric complexes. The complexes then translocate into the nucleus to regulate transcription of target genes. The TGF-β signaling pathway interacts with a variety of other intracellular signaling mechanisms [37]. For example, MAPK, NF-κB or PI3K/AKT pathways can either be activated by TGF-β, or can modulate the outcome of TGF-β induced Smad signaling [38, 39]. Cross-talk between TGF-β and other ligands, as well as the interaction of the Smad cascade with other intracellular signaling pathways add further complexity to these networks.

The development of colorectal cancers from normal epithelium is a multi-step process requiring inactivation of a series of tumor suppressor genes [40, 41] and the activation of specific oncogenes [42]. Normal human gut epithelium does not express endogenous GRPR or other BBS receptor subtypes [43]. However, a subset of human colorectal cancers expresses GRPR and its ligand GRP [43–45]. Activation of the GRPR by bombesin stimulates growth of colorectal cancer cells [46–49]. In this study we examined the cellular effects of activation of GRPR during colorectal carcinogenesis using RIE-1 cells stably transfected with GRPR [50]. RIE-1 cell line was selected for several reasons: (1) it is derived from rat small intestine and possesses the normal rat diploid number of chromosomes; it is a nontumorigenic and does not form colonies in soft agarose [51]; (2) unlike most human colonic cancer cell lines, it possesses a functional TGF-β signaling axis and responds to TGF-β-induced cell cycle arrest [11, 34] and apoptosis induction [17, 35]; (3) like the normal human colonic epithelium, it does not express endogenous GRPR or other BBS receptor subtypes [29]; (4) it has been used by many investigators to study the activation of oncogenes during colorectal carcinogenesis [30, 52, 53]. We found that RIE/GRPR cells did not develop transforming phenotypes such as colony formation in soft agarose (data not shown). Surprisingly, BBS inhibited cell growth and enhanced TGF-β’s growth inhibitory effects. In contrast, our previous study demonstrated that BBS enhanced TGF-β-induced COX-2 expression [29] that is associated with the tumor promoting effects of TGF-β [54]. There is substantial evidence to support the contention that TGF-β is a potent growth inhibitor with tumor-suppressing activity. As well, it has tumor promoting effects and contributes to tumor cell aggressiveness due to genetic loss of TGF-β signaling components or because of downstream perturbation of the signaling pathway [54, 55]. Taken together our studies demonstrate that GRPR enhances both the tumor suppressor function and tumor promoting effects of TGF-β, however, overexpression of GRPR alone is insufficient for colorectal carcinogenesis. Therefore, we speculate that activation of GRPR by bombesin promotes cell growth when the TGF-β tumor suppressor function is inactivated late in colorectal carcinogenesis. This novel hypothesis is currently being tested in our laboratory.

In conclusion, we have demonstrated in this study that BBS enhanced TGF-β-induced growth inhibition through induction of apoptosis, suggesting the interaction between TGF-β and GRPR signaling pathways responsible for functional consequences. Furthermore, p38MAPK partially mediated this interaction. Our study provides further evidence that growth factors and gastric peptides interact to regulate gut function, and sheds light on better understanding the complicated network in gut physiological and pathological development.

ACKNOWLEDGMENTS

We thank Dr. D. Song for technical support; M. Griffin in Flow Cytometry and Cell Sorting Core Facility, Univ. of Texas Medical Branch for flow cytometric analysis; E. Figueroa and S. Schuenke in the Department of Surgery, Univ. of Texas Medical Branch for manuscript preparation. This study was supported by Public Health Service grants P01 DK035608 (C.M.T. and T.C.K.) and R01 DK060105 (T.C.K.).

Footnotes

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REFERENCES

  • 1.Babyatsky MW, Podolsky DK. Growth and development in the gastrointestinal tract. In: Yamada T, Alpers DH, Owyang C, Powell DW, editors. Text book of Gastroenterology. Philadelphia: JB Lippincott Co.; 1991. pp. 475–501. [Google Scholar]
  • 2.Ko TC, Bresnahan WA, Thompson EA. Intestinal cell cycle regulation. In: Meijer L, Guidet S, Philippe M, editors. Progress in Cell Cycle Research. New York: Plenum; 1997. pp. 43–52. [DOI] [PubMed] [Google Scholar]
  • 3.Cairnie AB, Lamerton LF, Steel GG. Cell proliferation studies in the intestinal epithelium of the rat. I. Determination of the kinetic parameters. Exp Cell Res. 1965;39:528–538. doi: 10.1016/0014-4827(65)90055-8. [DOI] [PubMed] [Google Scholar]
  • 4.Potten CS, Wilson JW, Booth C. Regulation and significance of apoptosis in the stem cells of the gastrointestinal epithelium. Stem Cells. 1997;15:82–93. doi: 10.1002/stem.150082. [DOI] [PubMed] [Google Scholar]
  • 5.Hahm KB, Im YH, Parks TW, Park SH, Markowitz S, Jung HY, Green J, Kim SJ. Loss of transforming growth factor beta signalling in the intestine contributes to tissue injury in inflammatory bowel disease. Gut. 2001;49:190–198. doi: 10.1136/gut.49.2.190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Heldin CH, Miyazono K, ten Dijke P. TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature. 1997;390:465–471. doi: 10.1038/37284. [DOI] [PubMed] [Google Scholar]
  • 7.Massague J. TGF-beta signal transduction. Annu Rev Biochem. 1998;67:753–791. doi: 10.1146/annurev.biochem.67.1.753. [DOI] [PubMed] [Google Scholar]
  • 8.Massague J, Blain SW, Lo RS. TGFbeta signaling in growth control, cancer, and heritable disorders. Cell. 2000;103:295–309. doi: 10.1016/s0092-8674(00)00121-5. [DOI] [PubMed] [Google Scholar]
  • 9.Booth D, Haley JD, Bruskin AM, Potten CS. Transforming growth factor-B3 protects murine small intestinal crypt stem cells and animal survival after irradiation, possibly by reducing stem-cell cycling. Int J Cancer. 2000;86:53–59. doi: 10.1002/(sici)1097-0215(20000401)86:1<53::aid-ijc8>3.0.co;2-z. [DOI] [PubMed] [Google Scholar]
  • 10.Barnard JA, Beauchamp RD, Coffey RJ, Moses HL. Regulation of intestinal epithelial cell growth by transforming growth factor type beta. Proc Natl Acad Sci U S A. 1989;86:1578–1582. doi: 10.1073/pnas.86.5.1578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Ko TC, Sheng HM, Reisman D, Thompson EA, Beauchamp RD. Transforming growth factor-beta 1 inhibits cyclin D1 expression in intestinal epithelial cells. Oncogene. 1995;10:177–184. [PubMed] [Google Scholar]
  • 12.Smith RD. Evidence for autocrine growth inhibition of rat intestinal epithelial (RIE-1) cells by transforming growth factor type-beta. Biochem Mol Biol Int. 1995;35:1315–1321. [PubMed] [Google Scholar]
  • 13.Ciacci C, Lind SE, Podolsky DK. Transforming growth factor beta regulation of migration in wounded rat intestinal epithelial monolayers. Gastroenterology. 1993;105:93–101. doi: 10.1016/0016-5085(93)90014-4. [DOI] [PubMed] [Google Scholar]
  • 14.Wang JY, Viar MJ, Li J, Shi HJ, McCormack SA, Johnson LR. Polyamines are necessary for normal expression of the transforming growth factor-beta gene during cell migration. Am J Physiol. 1997;272:G713–G720. doi: 10.1152/ajpgi.1997.272.4.G713. [DOI] [PubMed] [Google Scholar]
  • 15.Kurokowa M, Lynch K, Podolsky DK. Effects of growth factors on an intestinal epithelial cell line: transforming growth factor beta inhibits proliferation and stimulates differentiation. Biochem Biophys Res Commun. 1987;142:775–782. doi: 10.1016/0006-291x(87)91481-1. [DOI] [PubMed] [Google Scholar]
  • 16.Dunker N, Schmitt K, Schuster N, Krieglstein K. The role of transforming growth factor beta-2, beta-3 in mediating apoptosis in the murine intestinal mucosa. Gastroenterology. 2002;122:1364–1375. doi: 10.1053/gast.2002.32991. [DOI] [PubMed] [Google Scholar]
  • 17.Cao Y, Chen L, Zhang W, Liu Y, Papaconstantinou HT, Bush CR, Townsend CM, Jr, Thompson EA, Ko TC. Identification of apoptotic genes mediating TGF-beta/Smad3-induced cell death in intestinal epithelial cells using a genomic approach. Am J Physiol Gastrointest Liver Physiol. 2007;292:G28–G38. doi: 10.1152/ajpgi.00437.2005. [DOI] [PubMed] [Google Scholar]
  • 18.Matsushita M, Matsuzaki K, Date M, Watanabe T, Shibano K, Nakagawa T, Yanagitani S, Amoh Y, Takemoto H, Ogata N, Yamamoto C, Kubota Y, Seki T, Inokuchi H, Nishizawa M, Takada H, Sawamura T, Okamura A, Inoue K. Down-regulation of TGF-beta receptors in human colorectal cancer: implications for cancer development. Br J Cancer. 1999;80:194–205. doi: 10.1038/sj.bjc.6690339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Thiagalingam S, Lengauer C, Leach FS, Schutte M, Hahn SA, Overhauser J, Willson JK, Markowitz S, Hamilton SR, Kern SE, Kinzler KW, Vogelstein B. Evaluation of candidate tumour suppressor genes on chromosome 18 in colorectal cancers. Nat Genet. 1996;13:343–346. doi: 10.1038/ng0796-343. [DOI] [PubMed] [Google Scholar]
  • 20.MacGrogan D, Pegram M, Slamon D, Bookstein R. Comparative mutational analysis of DPC4 (Smad4) in prostatic and colorectal carcinomas. Oncogene. 1997;15:1111–1114. doi: 10.1038/sj.onc.1201232. [DOI] [PubMed] [Google Scholar]
  • 21.Erspamer GF, Cei JM. Biogenic amines and active polypeptides in the skin of Leptodactylus vilarsi melin. Biochem Pharmacol. 1970;19:321–325. doi: 10.1016/0006-2952(70)90189-9. [DOI] [PubMed] [Google Scholar]
  • 22.McDonald TJ, Jornvall H, Nilsson G, Vagne M, Ghatei M, Bloom SR, Mutt V. Characterization of a gastrin releasing peptide from porcine non-antral gastric tissue. Biochem Biophys Res Commun. 1979;90:227–233. doi: 10.1016/0006-291x(79)91614-0. [DOI] [PubMed] [Google Scholar]
  • 23.Patel O, Shulkes A, Baldwin GS. Gastrin-releasing peptide and cancer. Biochim Biophys Acta. 2006;1766:23–41. doi: 10.1016/j.bbcan.2006.01.003. [DOI] [PubMed] [Google Scholar]
  • 24.Baroni A, Perfetto B, Canozo N, Braca A, Farina E, Melito A, De Maria S, Carteni M. Bombesin: a possible role in wound repair. Peptides. 2008;29:1157–1166. doi: 10.1016/j.peptides.2008.03.006. [DOI] [PubMed] [Google Scholar]
  • 25.Sun YG, Chen ZF. A gastrin-releasing peptide receptor mediates the itch sensation in the spinal cord. Nature. 2007;448:700–703. doi: 10.1038/nature06029. [DOI] [PubMed] [Google Scholar]
  • 26.Carroll RE, Matkowskyj K, Saunthararajah Y, Sekosan M, Battey JF, Benya RV. Contribution of gastrin-releasing peptide and its receptor to villus development in the murine and human gastrointestinal tract. Mech Dev. 2002;113:121–130. doi: 10.1016/s0925-4773(02)00032-1. [DOI] [PubMed] [Google Scholar]
  • 27.West SD, Mercer DW. Bombesin-induced gastroprotection. Ann Surg. 2005;241:227–231. doi: 10.1097/01.sla.0000151790.14274.5d. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Assimakopoulos SF, Scopa CD, Zervoudakis G, Mylonas PG, Georgiou C, Nikolopoulou V, Vagianos CE. Bombesin and neurotensin reduce endotoxemia, intestinal oxidative stress, and apoptosis in experimental obstructive jaundice. Ann Surg. 2005;241:159–167. doi: 10.1097/01.sla.0000149306.35717.8b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Guo YS, Chen Z, Wen XD, Ko TC, Townsend CM, Jr, Hellmich MR. Synergistic Regulation of COX-2 Expression by Bombesin and Transforming Growth Factor-beta. Dig Dis Sci. 2007 doi: 10.1007/s10620-007-0122-9. [DOI] [PubMed] [Google Scholar]
  • 30.Cao Y, Deng C, Townsend CM, Jr, Ko TC. TGF-beta inhibits Akt-induced transformation in intestinal epithelial cells. Surgery. 2006;140:322–329. doi: 10.1016/j.surg.2006.05.006. [DOI] [PubMed] [Google Scholar]
  • 31.Song S, Freedman J, Mody M, Lazarus AH. p38 MAPK is activated but not necessary in porcine von Willebrand factor-dependent platelet activation. Br J Haematol. 1999;107:532–538. doi: 10.1046/j.1365-2141.1999.01750.x. [DOI] [PubMed] [Google Scholar]
  • 32.Jin ZJ. [Addition in drug combination (author's transl)] Zhongguo Yao Li Xue Bao. 1980;1:70–76. [PubMed] [Google Scholar]
  • 33.Zhang KZ, Xu JH, Huang XW, Wu LX, Su Y, Chen YZ. Curcumin synergistically augments bcr/abl phosphorothioate antisense oligonucleotides to inhibit growth of chronic myelogenous leukemia cells. Acta Pharmacol Sin. 2007;28:105–110. doi: 10.1111/j.1745-7254.2007.00471.x. [DOI] [PubMed] [Google Scholar]
  • 34.Ko TC, Beauchamp RD, Townsend CM, Jr, Thompson EA, Thompson JC. Transforming growth factor-beta inhibits rat intestinal cell growth by regulating cell cycle specific gene expression. Am J Surg. 1994;167:14–19. doi: 10.1016/0002-9610(94)90048-5. discussion 9–20. [DOI] [PubMed] [Google Scholar]
  • 35.Cao Y, Liu X, Zhang W, Deng X, Zhang H, Liu Y, Chen L, Thompson EA, Townsend CM, Jr, Ko TC. TGF-beta repression of Id2 induces apoptosis in gut epithelial cells. Oncogene. 2009;28:1089–1098. doi: 10.1038/onc.2008.456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Papaconstatinou HT, Ko TC. Cell Cycle and Apoptosis Regulation in GI Cancers. In: Evers BM, editor. Molecular Mechanisms on Gastrointestinal Cancer. Austin: R.G. Landes Company; 1999. pp. 49–78. [Google Scholar]
  • 37.Javelaud D, Mauviel A. Crosstalk mechanisms between the mitogen-activated protein kinase pathways and Smad signaling downstream of TGF-beta: implications for carcinogenesis. Oncogene. 2005;24:5742–5750. doi: 10.1038/sj.onc.1208928. [DOI] [PubMed] [Google Scholar]
  • 38.Lutz M, Knaus P. Integration of the TGF-beta pathway into the cellular signalling network. Cell Signal. 2002;14:977–988. doi: 10.1016/s0898-6568(02)00058-x. [DOI] [PubMed] [Google Scholar]
  • 39.Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature. 2003;425:577–584. doi: 10.1038/nature02006. [DOI] [PubMed] [Google Scholar]
  • 40.Kinzler KW, Vogelstein B. Lessons from hereditary colorectal cancer. Cell. 1996;87:159–170. doi: 10.1016/s0092-8674(00)81333-1. [DOI] [PubMed] [Google Scholar]
  • 41.White RL. Tumor suppressing pathways. Cell. 1998;92:591–592. doi: 10.1016/s0092-8674(00)81124-1. [DOI] [PubMed] [Google Scholar]
  • 42.Cartwright CA, Meisler AI, Eckhart W. Activation of the pp60c-src protein kinase is an early event in colonic carcinogenesis. Proc Natl Acad Sci U S A. 1990;87:558–562. doi: 10.1073/pnas.87.2.558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Preston SR, Woodhouse LF, Jones-Blackett S, Miller GV, Primrose JN. High-affinity binding sites for gastrin-releasing peptide on human colorectal cancer tissue but not uninvolved mucosa. Br J Cancer. 1995;71:1087–1089. doi: 10.1038/bjc.1995.210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Radulovic SS, Milovanovic SR, Cai RZ, Schally AV. The binding of bombesin and somatostatin and their analogs to human colon cancers. Proc Soc Exp Biol Med. 1992;200:394–401. doi: 10.3181/00379727-200-43447. [DOI] [PubMed] [Google Scholar]
  • 45.Carroll R, Matkowskyj K, Chakrabarti S, McDonald T, Benya R. Aberrant expression of gastrin-releasing peptide and its receptor by well-differentiated colon cancers in humans. Am.J.Physiol. 1999;276:G655–G665. doi: 10.1152/ajpgi.1999.276.3.G655. [DOI] [PubMed] [Google Scholar]
  • 46.Radulovic S, Miller G, Schally AV. Inhibition of growth of HT-29 human colon cancer xenografts in nude mice by treatment with bombesin/gastrin releasing peptide antagonist (RC- 3095) Cancer Res. 1991;51:6006–6009. [PubMed] [Google Scholar]
  • 47.Radulovic S, Schally AV, Reile H, Halmos G, Szepeshazi K, Groot K, Milovanovic S, Miller G, Yano T. Inhibitory effects of antagonists of bombesin/gastrin releasing peptide (GRP) and somatostatin analog (RC-160) on growth of HT-29 human colon cancers in nude mice. Acta Oncol. 1994;33:693–701. doi: 10.3109/02841869409121784. [DOI] [PubMed] [Google Scholar]
  • 48.Narayan S, Guo YS, Townsend CM, Jr, Singh P. Specific binding and growth effects of bombesin-related peptides on mouse colon cancer cells in vitro. Cancer Res. 1990;50:6772–6778. [PubMed] [Google Scholar]
  • 49.Casanueva FF, Perez FR, Casabiell X, Camina JP, Cai RZ, Schally AV. Correlation between the effects of bombesin antagonists on cell proliferation and intracellular calcium concentration in Swiss 3T3 and HT-29 cell lines. Proc Natl Acad Sci U S A. 1996;93:1406–1411. doi: 10.1073/pnas.93.4.1406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Guo YS, Hellmich MR, Wen XD, Townsend CM., Jr Activator protein-1 transcription factor mediates bombesin-stimulated cyclooxygenase-2 expression in intestinal epithelial cells. J Biol Chem. 2001;276:22941–22947. doi: 10.1074/jbc.M101801200. [DOI] [PubMed] [Google Scholar]
  • 51.Blay J, Brown KD. Characterization of an epithelioid cell line derived from rat small intestine: demonstration of cytokeratin filaments. Cell Biol Int Rep. 1984;8:551–560. doi: 10.1016/0309-1651(84)90054-7. [DOI] [PubMed] [Google Scholar]
  • 52.Sheng H, Shao J, DuBois RN. Akt/PKB activity is required for Ha-Ras-mediated transformation of intestinal epithelial cells. J Biol Chem. 2001;276:14498–14504. doi: 10.1074/jbc.M010093200. [DOI] [PubMed] [Google Scholar]
  • 53.Yu W, Murray NR, Weems C, Chen L, Guo H, Ethridge R, Ceci JD, Evers BM, Thompson EA, Fields AP. Role of cyclooxygenase 2 in protein kinase C beta II-mediated colon carcinogenesis. J Biol Chem. 2003;278:11167–11174. doi: 10.1074/jbc.M211424200. [DOI] [PubMed] [Google Scholar]
  • 54.Roman C, Saha D, Beauchamp R. TGF-beta and colorectal carcinogenesis. Microsc Res Tech. 2001;52:450–457. doi: 10.1002/1097-0029(20010215)52:4<450::AID-JEMT1030>3.0.CO;2-O. [DOI] [PubMed] [Google Scholar]
  • 55.Akhurst RJ, Derynck R. TGF-beta signaling in cancer--a double-edged sword. Trends Cell Biol. 2001;11:S44–S51. doi: 10.1016/s0962-8924(01)02130-4. [DOI] [PubMed] [Google Scholar]

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