TGF-beta repression of Id2 induces apoptosis in gut epithelial cells.

Transforming growth factor-beta (TGF-beta) regulates epithelial tissue homeostasis by activating processes that control cell cycle arrest, differentiation and apoptosis. Disruption of the TGF-beta signaling pathway often occurs in colorectal cancers. Earlier, we have shown that TGF-beta induces apoptosis through the transcription factor Smad3. Affymetrix oligonucleotide microarrays were used to identify TGF-beta/Smad3 target genes that regulate apoptosis in rat intestinal epithelial cells (RIE-1). We found that TGF-beta repressed the expression of the inhibitor of differentiation (Id) gene family. Knockdown of Id1 and Id2 gene expression induced apoptosis in RIE-1 cells, whereas overexpression of Id2 attenuated TGF-beta-induced apoptosis. TranSignal Protein/DNA arrays were used to identify the hypoxia-inducing factor-1 (HIF-1) as a downstream target of TGF-beta. HIF-1 is a basic helix-loop-helix protein, and overexpression of Id2 blocked HIF-1 activation by TGF-beta. Furthermore, knockdown of HIF-1 blocked TGF-beta-induced apoptosis. Thus, we have identified HIF-1 as a novel mediator downstream of Id2 in the pathway of TGF-beta-induced apoptosis.

Constitutively active CCK2 receptor splice variant increases Src-dependent HIF-1 alpha expression and tumor growth.

Gastrointestinal (GI) cancers ectopically express multiple splice variants of the cholecystokinin-2 (CCK(2))/gastrin receptor; however, their relative contributions to the cancer phenotype are unknown. The aim of this study was to compare the effects of CCK(2) receptor (CCK(2)R) and CCK(2i4sv)R expression on cell growth both in vitro and in vivo using a human epithelial cell model, HEK239. In vitro, receptor variant expression did not affect cell proliferation either in the absence or presence of agonist. However, in vivo, the expression of CCK(2i4sv)R, but not CCK(2)R, increases HEK293 tumor growth in a constitutive, Src-dependent manner. Enhanced tumorigenicity of CCK(2i4sv)R is associated with an Src-dependent increase in the transcription factor, hypoxia-inducible factor-1alpha, its downstream target, vascular endothelial growth factor and tumor micro-vessel density, suggesting that CCK(2i4sv)R may contribute to the growth and spread of GI cancers through agonist-independent mechanisms that enhance tumor angiogenesis.

Phosphatidylinositol 3-kinase mediates proliferative signals in intestinal epithelial cells.

BACKGROUND AND AIMS: Determination of intracellular signalling pathways that mediate intestinal epithelial proliferation is fundamental to the understanding of the integrity and function of the intestinal tract under normal and diseased conditions. The phosphoinositide 3-kinase (PI3K)/Akt pathway transduces signals initiated by growth factors and is involved in cell proliferation and differentiation. In this study, we assessed the role of PI3K/Akt in transduction of proliferative signals in intestinal epithelial cells. METHODS: A rat intestinal epithelial (RIE) cell line and human colorectal cancer HCA-7 and LS-174 cell lines served as in vitro models. The Balb/cJ mouse was the in vivo model. RESULTS: PI3K activation was critical for G1 cell cycle progression of intestinal epithelial cells. Ectopic expression of either active p110alpha or Akt-1 increased RIE cell proliferation. In vivo experiments demonstrated that PI3K activation was closely associated with the proliferative activity of intestinal mucosa. Treatment of mice with PI3K inhibitors blocked induction of PI3K activity and attenuated intestinal mucosal proliferation associated with oral intake. Epidermal growth factor and transforming growth factor alpha stimulated PI3K activation which was required for growth factor induced expression of cyclin D1. CONCLUSIONS: The PI3K/Akt pathway transduces mitogenic signals from growth factor receptors to the cell cycle machinery and plays a critical role in regulation of intestinal epithelial proliferation.

The role of caspases in methotrexate-induced gastrointestinal toxicity.

BACKGROUND: Enterocolitis is the major toxicity of methotrexate-based cancer chemotherapy, which limits its clinical applications. Methotrexate induces gut mucosal apoptosis in vivo; however, little is known about the molecular mechanism involved. The effectors of apoptosis include the caspase family of proteases, which are selectively activated in a stimulus-specific and tissue-specific fashion. The aims of this study were (1) to establish an in vitro model of methotrexate-induced gut apoptosis and (2) to determine the role of caspases in methotrexate-induced apoptosis in intestinal epithelial cells. METHODS: Rat intestinal epithelial cells (RIE-1) were treated with methotrexate in the absence or presence of ZVAD-fluoromethyl ketone, a general caspase inhibitor. Apoptosis was quantified by means of deoxyribonucleic acid (DNA) fragmentation assays and Hoechst nuclear staining. Caspase activation was measured with the use of fluorogenic substrates. RESULTS: Methotrexate induced apoptosis and decreased cell number in RIE-1 cells. DNA fragmentation was preceded by the sequential activation of caspases 9, 2, and 3, whereas caspases 1 and 8 remained inactive. ZVAD-fluoromethyl ketone inhibited methotrexate-induced caspase activation, DNA fragmentation, and nuclear condensation. CONCLUSIONS: These results indicate that methotrexate activates specific caspases and induces apoptosis in RIE-1 cells. Furthermore, caspases may play an important role in methotrexate-induced apoptosis in RIE-1 cells and may be potential therapeutic targets to attenuate methotrexate-induced enterocolitis.

Amino acid, glucose, and lipid kinetics after palliative resection in a patient with glucagonoma syndrome.

Glucagon excess causes catabolic changes, including enhanced glucose production, lipolysis, and amino acid oxidation. In this study, we evaluate the metabolic effects of debulking surgery on a patient with glucagon-producing tumor. Stable isotope tracer methods were used to measure glucose, glycerol, and alpha-ketoisocaproic acid (alpha KICA) rates of appearance (Ra) into plasma. Measurements were obtained 25 days after surgery in the basal state and during hormonal suppression of glucagon production by infusing somatostatin with insulin replacement. Basal plasma glucagon concentration (14,100 pg/mL) remained high after debulking surgery. Somatostatin infusion decreased plasma glucagon concentration to 6,735 pg/mL and basal substrate kinetics (alpha-KICA Ra from 1.97 to 1.48 micromol/kg/min; glucose Ra from 16.89 to 11.56 micromol/kg/min; and glycerol Ra from 3.33 to 2.74 micromol/kg/min). We conclude that debulking surgery fails to adequately suppress glucagon production and the alterations in substrate metabolism associated with excess glucagon. In these patients, somatostatin therapy can be an effective method to suppress secretion of glucagon and help attenuate its catabolic effects.

Activator protein-1 transcription factor mediates bombesin-stimulated cyclooxygenase-2 expression in intestinal epithelial cells.

Colorectal carcinogenesis is a complex, multistep process involving genetic alterations and progressive changes in signaling pathways regulating intestinal epithelial cell proliferation, differentiation, and apoptosis. Although cyclooxygenase-2 (COX-2), gastrin-releasing peptide (GRP), and its receptor, GRP-R, are not normally expressed by the epithelial cells lining the human colon, the levels of all three proteins are aberrantly overexpressed in premalignant adenomatous polyps and colorectal carcinomas of humans. Overexpression of these proteins is associated with altered epithelial cell growth, adhesion, and tumor cell invasiveness, both in vitro and in vivo; however, a mechanistic link between GRP-R-mediated signaling pathways and increased COX-2 overexpression has not been established. We report that bombesin, a homolog of GRP, potently stimulates the expression of COX-2 mRNA and protein as well as the release of prostaglandin E(2) from a rat intestinal epithelial cell line engineered to express GRP-R. Bombesin stimulation of COX-2 expression requires an increase in [Ca(2+)](i), activation of extracellular signal-regulated kinase (ERK)-1 and -2 and p38(MAPK), and increased activation and expression of the transcription factors Elk-1, ATF-2, c-Fos, and c-Jun. These data suggest that the expression of GRP-R in intestinal epithelial cells may play a role in carcinogenesis by stimulating COX-2 overexpression through an activator protein-1-dependent pathway.

Prevention of mucosal atrophy: role of glutamine and caspases in apoptosis in intestinal epithelial cells.

Glutamine starvation induces apoptosis in enterocytes; therefore glutamine is important in the maintenance of gut mucosal homeostasis. However, the molecular mechanisms are unknown. The caspase family of proteases constitutes the molecular machinery that drives apoptosis. Caspases are selectively activated in a stimulus-specific and tissue-specific fashion. The aims of this study were to (1) identify specific caspases activated by glutamine starvation and (2) determine whether a general caspase inhibitor blocks glutamine starvation-induced apoptosis in intestinal epithelial cells. Rat intestinal epithelial (RIE-1) cells were deprived of glutamine. Specific caspase activation was measured using fluorogenic substrate assay. Apoptosis was quantified by DNA fragmentation and Hoechst nuclear staining. Glutamine starvation of RIE-1 cells resulted in the time-dependent activation of caspases 3 (10 hours) and 2 (18 hours), and the induction of DNA fragmentation (12 hours). Caspases 1 and 8 remained inactive ZVAD-fluoromethyl ketone, a general caspase inhibitor, completely blocked glutamine starvation-induced caspase activation, DNA fragmentation, and nuclear condensation. These results indicate that glutamine starvation selectively activates specific caspases, which leads to the induction of apoptosis in RIE-1 cells. Furthermore, inhibition of caspase activity blocked the induction of apoptosis, suggesting that caspases are potential molecular targets to attenuate apoptotic responses in the gut.

Roles of gastrointestinal hormones in pancreatic cancer.

Several gastrointestinal (GI) hormones, such as gastrin, cholecystokinin, and bombesin, have been reported to affect the development of pancreatic cancer. The receptors for these hormones are found in normal and neoplastic pancreatic cells. Activation of these receptors enhances pancreatic carcinogenesis and promotes the growth of established pancreatic carcinoma either in vitro or in vivo. On the other hand, some studies have shown that these GI hormones may have no effect or may play an inhibitory role in the development of pancreatic cancer. The reasons for the apparent discrepancies in the published literature are discussed in this review. In recent years, increasing emphasis has been placed on the effects of GI hormones on cancer invasion and metastasis. As the transition from noninvasion to the invasive state is the crucial event in cancer development, further investigation of the way in which GI hormones affect the invasion and metastasis of pancreatic cancer may be important for the development of new therapeutic approaches with eventual clinical utility.

Signaling mechanisms regulating bombesin-mediated AP-1 gene induction in the human gastric cancer SIIA.

The hormone bombesin (BBS) and its mammalian equivalent gastrin-releasing peptide (GRP) act through specific GRP receptors (GRP-R) to affect multiple cellular functions in the gastrointestinal tract; the intracellular signaling pathways leading to these effects are not clearly defined. Previously, we demonstrated that the human gastric cancer SIIA possesses GRP-R and that BBS stimulates activator protein-1 (AP-1) gene expression. The purpose of our present study was to determine the signaling pathways leading to AP-1 induction in SIIA cells. A rapid induction of c-jun and jun-B gene expression was noted after BBS treatment; this effect was blocked by specific GRP-R antagonists, indicating that BBS is acting through the GRP-R. The signaling pathways leading to increased AP-1 gene expression were delineated using phorbol 12-myristate 13-acetate (PMA), which stimulates protein kinase C (PKC)-dependent pathways, by forskolin (FSK), which stimulates protein kinase A (PKA)-dependent pathways, and by the use of various protein kinase inhibitors. Treatment with PMA stimulated AP-1 gene expression and DNA binding activity similar to the effects noted with BBS; FSK stimulated jun-B expression but produced only minimal increases of c-jun mRNA and AP-1 binding activity. Pretreatment of SIIA cells with either H-7 or H-8 (primarily PKC inhibitors) inhibited the induction of c-jun and jun-B mRNAs in response to BBS, whereas H-89 (PKA inhibitor) exhibited only minimal effects. Pretreatment with tyrphostin-25, a protein tyrosine kinase (PTK) inhibitor, attenuated the BBS-mediated induction of c-jun and jun-B, but the effect was not as pronounced as with H-7. Collectively, our results demonstrate that BBS acts through its receptor to produce a rapid induction of both c-jun and jun-B mRNA and AP-1 DNA binding activity in the SIIA human gastric cancer. Moreover, this induction of AP-1, in response to BBS, is mediated through both PKC- and PTK-dependent signal transduction pathways with only minimal involvement of PKA.

Human colorectal cancers express a constitutively active cholecystokinin-B/gastrin receptor that stimulates cell growth.

Although ectopic expression of the cholecystokinin B/gastrin receptor (CCK-BR) is widely reported in human colorectal cancers, its role in mediating the proliferative effects of gastrin1-17 (G-17) on these cancers is unknown. Here we report the isolation of a novel splice variant of CCK-BR that exhibits constitutive (ligand-independent) activation of pathways regulating intracellular free Ca(2+) ([Ca(2+)](i)) and cell growth. The splice variant (designated CCK-BRi4sv for intron 4-containing splice variant) is expressed in colorectal cancers but not in normal colonic mucosa adjacent to the cancer. Balb3T3 cells expressing CCK-BRi4sv exhibited spontaneous, ligand-independent, oscillatory increases in [Ca(2+)](i), whereas cells expressing wild-type CCK-BR did not. Primary cultures of cells isolated from resected colorectal cancers also exhibited a similar pattern of spontaneous [Ca(2+)](i) oscillations. For both Balb3T3 and primary tumor cells, application of G-17 (10 and 200 nm, respectively) caused an increase in [Ca(2+)](i). Selective CCK-BR antagonists blocked the G-17-stimulated Ca(2+) responses but not the spontaneous [Ca(2+)](i) oscillations. Cells expressing CCK-BRi4sv exhibited an increased growth rate ( approximately 2.5-fold), in the absence of G-17, compared with cells expressing wild-type CCK-BR. The selective pattern of expression, constitutive activity, and trophic action associated with CCK-BRi4sv suggest that this variant may regulate colorectal cancer cell proliferation though a gastrin-independent mechanism.

Gut peptide receptor expression in human pancreatic cancers.

OBJECTIVE: To determine the prevalence of gastrointestinal (GI) peptide receptor expression in pancreatic cancers, and to further assess signaling mechanisms regulating neurotensin (NT)-mediated pancreatic cancer growth. SUMMARY BACKGROUND DATA: Pancreatic cancer remains one of the leading causes of GI cancer death; novel strategies for the early detection and treatment of these cancers is required. Previously, the authors have shown that NT, an important GI hormone, stimulates the proliferation of an NT receptor (NTR)-positive pancreatic cancer. METHODS: A total of 26 human pancreatic adenocarcinomas, obtained after resection, and 5 pancreatic cancer xenografts were analyzed for expression of NTR, vasoactive intestinal peptide receptor (VIPR), substance P receptor (SPR), and gastrin-releasing peptide receptor (GRPR). In addition, NTR expression, [Ca2+]i mobilization, and growth in response to NT was determined in L3.6, a metastatic pancreatic cancer cell line. RESULTS: Neurotensin receptor was expressed in 88% of the surgical specimens examined and all five of the pancreatic cancer xenografts. In contrast, VIPR, SPR, and GRPR expression was detected in 31%, 27%, and 8% of pancreatic cancers examined, respectively. Expression of NTR, functionally coupled to the Ca2+ signaling pathway, was identified in L3.6 cells; treatment with NT (10 micromol/L) stimulated proliferation of these cells. CONCLUSIONS: The authors demonstrated NTR expression in most of the pancreatic adenocarcinomas examined. In contrast, VIPR, SPR, and GRPR expression was detected in fewer of the pancreatic cancers. The expression of NTR and other peptide receptors suggests the potential role of endocrine manipulation in the treatment of these cancers. Further, the presence of GI receptors may provide for targeted chemotherapy or radiation therapy or in vivo scintigraphy for early detection.

Induction of apoptosis in human gastric cancer by sodium butyrate.

BACKGROUND: Novel therapeutic agents are needed in the adjuvant treatment of gastric cancer. The differentiating agent sodium butyrate (NaBT) inhibits the growth of colon cancer cells; its effects on gastric cancers are not known. The purpose of our study was to characterize the effects of NaBT on human gastric cancer. MATERIAL & METHODS: The human gastric cancer, SIIA, was treated with NaBT (5 mM) for 12-72 h. Cell number, viability and death were measured. Expression levels of the tumor-suppressor protein, p53, the cell-cycle inhibitors, p21Waf1/Cip1 and p27Kip1, and the pro-apoptotic proteins, Bax, Bak, and Bik, were determined. RESULTS: NaBT significantly inhibited SIIA gastric cancer cell proliferation in a time-dependent fashion by a process involving the induction of apoptosis. Treatment with NaBT was associated with increased expression levels of p21Waf1/Cip1 p27Kip1, Bax, Bak, and Bik. CONCLUSIONS: NaBT triggers growth arrest and apoptosis in the human gastric cancer SIIA potentially through the induction of the cell-cycle inhibitors, p21Waf1/Cip1 and p27Kip1, and the proapoptotic genes, Bax, Bak, and Bik. NaBT may be an effective adjuvant agent in the treatment of gastric cancer.

Targeting molecular pathways with camptothecin as novel therapy for gastric cancer.

Novel chemotherapeutic agents are needed to treat gastric cancer for which the prognosis remains dismal. The antitumor alkaloid camptothecin (CPT) may be useful in the treatment of certain solid tumors; however, its effects on gastric cancer are largely undefined. The purpose of our study was to characterize the effects of CPT on human gastric tumors in vivo and to determine the cellular mechanisms involved in CPT-mediated inhibition. Two human gastric cancers, WIL and TOR, were transplanted subcutaneously into athymic nude mice. After tumors reached 50 to 100 mm(2), mice were randomized into three groups to receive injections of either low-dose CPT (5 mg/kg), high-dose CPT (10 mg/kg), or vehicle (control) intraperitoneally 3 days a week for 3 weeks. Tumors were measured and weighed, and protein levels of the cell cycle inhibitor, p21Waf1/Cip1, and the antiapoptotic protein, Bcl-2, were assessed. Both dosages of CPT significantly inhibited growth of WIL and TOR gastric tumors. CPT (10 mg/kg) reduced tumor size compared to baseline, establishing this as a tumoricidal dosage. Treatment with CPT was associated with increased levels of p21Waf1/Cip1 and decreased levels of Bcl-2. CPT effectively kills human gastric cancers associated with increased levels of p21Waf1/Cip1 and decreased levels of Bcl-2. By activating cell cycle withdrawal and cell death through induction of p21Waf1/Cip1 and downregulation of Bcl-2, CPT may be an effective agent for gastric cancer.

Enterotrophic effects of glucagon-like peptide 2 are enhanced by neurotensin.

Combination therapy with enterotrophic agents may be useful in patients with the short bowel syndrome. The gut hormones neurotensin (NT) and glucagon-like peptide 2 (GLP-2) are potent enterotrophic factors when administered alone; however, their combined effects are not known. Using a GLP-2-producing tumor (STC-1), we determined whether administration of NT enhances the effect of GLP-2 on intestinal growth. Athymic mice were injected with STC-1 cells (6 x 10(6)) subcutaneously. Twenty-three days after STC-1 implantation, mice received either NT (300 microg/kg or 600 microg/kg) or saline solution (control) subcutaneously three times a day for 6 days. Two groups of tumor-free mice received either saline or NT for 6 days. At sacrifice, jejunum and ileum were collected, weighed, and analyzed for DNA and protein content. In the jejunum, NT combined with GLP-2 (from STC-1) increased weight, protein content (markers of mucosal hypertrophy), and DNA content (a marker of mucosal hyperplasia), compared to either NT or GLP-2 alone. In the ileum, the combination of NT and GLP-2 significantly increased weight and/or protein content compared to NT or GLP-2 alone. Administration of NT enhances the enterotrophic effects of GLP-2, augmenting hypertrophy of the entire small bowel and hyperplasia of the jejunum. The combination of NT and GLP-2 may be useful to enhance intestinal growth in patients with the short bowel syndrome.

Inhibition of gastric cancer by camptothecin involves apoptosis and multiple cellular pathways.

BACKGROUND: The prognosis for gastric cancer remains dismal; novel agents that target specific molecular pathways are needed as adjuvant therapy. Camptothecin (CPT), on inhibitor of topoisomerase I, is effective in the treatment of certain solid tumors; its effects on gastric cancer are largely undefined. The purpose of this study was to (1) characterize the effects of CPT on the growth of a human gastric cancer and (2) assess potential cellular mechanisms responsible for CPT-mediated growth inhibition. METHODS: The human gastric cancer SIIA was transplanted subcutaneously into athymic nude mice. After tumors reached approximately 100 mm2, mice were randomized into 3 groups to receive either CPT (5 or 10 mg/kg) or vehicle (control) intraperitoneally 3 days per week for 3 weeks; tumor size was measured biweekly. To assess potential mechanisms of CPT-mediated inhibition, SIIA cells were treated with CPT (20 mumol/L) and cells were counted over a time course; apoptosis was assessed by Hoechst stain and DNA laddering. Expression of p53 (a tumor suppressor), p21Waf1 and p27Kip1 (cell cycle inhibitors), and Bcl-2 and Bcl-XL (antiapoptotic proteins) was determined. RESULTS: CPT (5 and 10 mg/kg) significantly inhibited tumor growth of SIIA gastric cancers compared with controls. CPT-mediated inhibition of SIIA cell proliferation was associated with an increase in apoptosis. Moreover, CPT treatment resulted in induction of p53, p21Waf1, and p27Kip1 and a decrease in Bcl-2 and Bcl-XL RNA and protein levels. CONCLUSIONS: Treatment with CPT effectively inhibited the growth of the human gastric cancer SIIA; the mechanism involved was induction of apoptosis mediated by up-regulation of p53, p21Waf1/Cip1, and p27Kip1 and the down-regulation of Bcl-2 and Bcl-XL. Novel agents such as CPT, which target specific molecular pathways, may prove clinically useful in the adjuvant treatment of gastric cancers.

JMV1155: a novel inhibitor of glycine-extended progastrin-mediated growth of a human colon cancer in vivo.

BACKGROUND: Glycine-extended progastrin (G-17-Gly), the immediate biosynthetic precursor to gastrin (G-17), stimulates growth of some gastrointestinal cancers in vitro. The purpose of this study was twofold: to evaluate the effects of G-17-Gly on a human colon cancer (DLD-1) in vivo and to determine whether the novel gastrin-receptor antagonist, JMV1155, inhibits G-17-Gly-mediated growth. METHODS: DLD-1 cells (2 x 10(6)) were injected subcutaneously (s.c.) at a single site in athymic nude mice. Mice were randomized to four groups (n = 6/group) to receive injections, s.c., tid of either saline (control), G-17-Gly, JMV1155, or G-17-Gly + JMV1155 for 28 days. Tumors were measured biweekly until sacrifice at which time tumors were weighed and analyzed for DNA and protein content. RESULTS: JMV1155 significantly inhibited G-17-Gly-stimulated growth of DLD-1 tumors by 14 days of treatment, producing a 56% decrease in tumor size by 28 days. JMV1155 also significantly decreased G-17-Gly-mediated increases in tumor weight (by 64%), DNA content (by 61%), and protein content (by 65%). CONCLUSIONS: We have demonstrated, for the first time, that the novel gastrin-receptor antagonist, JMV1155, blocks G-17-Gly-induced growth of a transplanted human colon cancer in vivo. Hormonally based therapy with JMV1155 potentially could be employed for some patients with colon carcinoma.

Characterization of two novel proabsorptive peptide YY analogs, BIM-43073D and BIM-43004C.

Effective clinical therapy to augment intestinal absorption of water and electrolytes does not exist; the gut hormone, peptide YY (PYY), is a potent proabsorptive agent in animal models. The purpose of our study was to evaluate the effects of two novel PYY analogs, BIM-43073D and BIM-43004C, on intestinal absorption. Dogs with ileal Thiry-Vella fistulae (TVF) were treated with either PYY, BIM-43073D, or BIM-43004C. Administration of BIM-43073D significantly increased water and sodium absorption over baseline and maintained this level of increased absorption for a longer duration than an equimolar dose of PYY. Administration of BIM-43004C significantly increased sodium and water absorption over baseline at a level equal to that of PYY. The novel PYY analogs, BIM-43073D and BIM-43004C, are effective proabsorptive agents with BIM-43073D producing more sustained effects than PYY. These compounds may be clinically useful in the treatment of gut malabsorption in conditions such as cholera, Crohn's disease, and the short-bowel syndrome.

Glucagon-like peptide 2 is a potent growth factor for small intestine and colon.

Factors that stimulate gut mucosal proliferation may be beneficial during periods of gut disuse or atrophy. Recently glucagon-like peptide 2 (GLP-2) has been shown to stimulate small bowel growth. The purpose of our study was to compare the trophic effects of GLP-2 with those of neurotensin (NT), a potent gut trophic factor. Mice were randomized to receive either GLP-2, NT, or saline solution (control) for 10 days. The mice were killed on day 11, at which time the jejunum, ileum, and colon were removed, weighed, and DNA and protein content measured. Mice treated with GLP-2 showed a significant increase in the weight of the jejunum, ileum, and colon compared to both control and NT-treated mice. DNA content, a marker of cellular hyperplasia, was significantly increased in the small bowel and colon by treatment with GLP-2 and NT compared to control tissues. Small intestinal protein content, an indicator of cellular hypertrophy, was significantly increased by GLP-2 compared to both NT and control; protein content of the colon was greater in each of the treatment groups compared with control mice. We have demonstrated, for the first time, that GLP-2 stimulates colonic growth. In addition, GLP-2 is a potent trophic factor of normal small intestine with proliferative effects that are equal to or greater than those of NT. Administration of GLP-2 may be useful clinically to enhance small intestinal regeneration and adaptation during periods of disease and in the early phases of the short bowel syndrome.

A novel cytotoxic agent for human carcinoid tumors.

BACKGROUND: Conventional adjuvant therapy for advanced carcinoid tumors remains disappointing; novel therapeutic agents are needed. We have shown previously that inhibiting polyamine biosynthesis with alpha-difluoromethylornithine (DFMO) slows the growth of carcinoid tumors. However, the clinical utility of DFMO has been limited by its cytostatic property. Synthetic polyamine analogs such as 1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) appear to be cytotoxic against several human tumors. The purpose of our study was to determine whether BE-4-4-4-4 is a more effective antiproliferative and cytotoxic agent than DFMO on human carcinoid (BON) cells in vitro. METHODS: BON cells were treated with either 5 mmol/L DFMO, 0.5 to 10 mumol/L BE-4-4-4-4, or vehicle (control). Ornithine decarboxylase activity was determined by the rate of 14CO2 production, and intracellular polyamine levels were determined by chromatography. Cell number and viability were determined by Coulter counter and trypan blue exclusion, respectively. RESULTS: BE-4-4-4-4 inhibited ornithine decarboxylase activity and depleted all 3 polyamines. BE-4-4-4-4 decreased cell numbers by 81% compared with control and 27% compared with DFMO. BE-4-4-4-4 also induced a 2-fold increase in cell death compared with control or DFMO. CONCLUSIONS: BE-4-4-4-4 is cytotoxic and more effective than DFMO in inhibiting growth of BON cells. Polyamine analogs such as BE-4-4-4-4 may be effective adjuvant therapeutic agents for advanced carcinoid tumors.

A novel strategy for inhibiting growth of human pancreatic cancer cells by blocking cyclin-dependent kinase activity.

Pancreatic cancers frequently carry mutations in the K-ras, p53, and p16 genes, which regulate cell proliferation. Transition from G1 to S phase of the cell cycle requires activation of cyclin-dependent kinase 2 (Cdk2) which is inhibited by olomoucine and roscovitine. The purpose of this study was to determine whether olomoucine and roscovitine can block Cdk2 kinase activity and inhibit proliferation of four human pancreatic cancer cell lines with various genetic alterations. Human pancreatic carcinoma cell lines BxPC-3, PANC-1 Capan-2, and CAV were treated with olomoucine or roscovitine. Cdk2 kinase activity was determined using histone H1 as the substrate. Cell cycle distribution was analyzed by DNA flow cytometry. Cell numbers were quantitated by Coulter counter. Olomoucine and roscovitine blocked Cdk2 activity in all four pancreatic cancer cell lines. Both compounds also inhibited cell proliferation in a dose-dependent fashion. Roscovitine was at least threefold more potent than olomoucine for both Cdk2 activity and cell proliferation. We have shown that Cdk inhibitors, olomoucine and roscovitine, block proliferation of human pancreatic cancer cells regardless of their mutations in K-ras p53, or p16 genes. These compounds represent a novel therapeutic strategy with potential therapeutic benefits for pancreatic cancers.