Glucose-dependent insulinotropic polypeptide monoclonal antibodies prevent and treat obesity in wild-type and hyperphagic mice.

OBJECTIVE: This study aimed to investigate whether a glucose-dependent insulinotropic polypeptide (GIP) monoclonal antibody (mAb) will promote weight loss in wild-type mice and to determine effects of this mAb in preventing weight gain in ob/ob mice. METHODS: Phosphate-buffered saline (PBS) or GIP mAb was injected intraperitoneally to wild-type mice fed a 60% high-fat diet (HFD). After 12 weeks, mice that received PBS were divided into two groups and were fed a 37% HFD for 5 weeks; one group received PBS, and one group received GIP mAb. In a separate study, PBS or GIP mAb was injected intraperitoneally to ob/ob mice fed normal mouse chow for 8 weeks. RESULTS: PBS-treated mice gained significantly more than those treated with GIP mAb, with no difference in food consumption detected. Obese mice fed a 37% HFD and PBS continued to gain weight (+2.1% ± 0.9%), whereas mice administered GIP mAb lost 4.1% ± 1.4% body weight (p < 0.01). Leptin-deficient mice consumed similar amounts of chow, and, after 8 weeks, the PBS- and GIP mAb-treated mice gained 250.4% ± 9.1% and 192.4% ± 7.3%, respectively (p < 0.01). CONCLUSIONS: These studies support the hypothesis that a reduction in GIP signaling appears to affect body weight without suppressing food intake and might provide a novel, useful method for the treatment and prevention of obesity.

Effects of gastric inhibitory polypeptide (GIP) immunoneutralization on mouse motor coordination and memory.

Gastric inhibitory polypeptide (GIP) is a regulatory peptide expressed in the mammalian upper small intestine, and both GIP and its receptor (GIPR) are expressed in the cortex and hippocampus regions of the brain as well. While learning and memory deficits have been observed in GIPR-/- mice, the effects of peripheral GIP immunoneutralization on motor-coordination, learning, and memory have not been examined. In the present study, adult GIPR-/- mice (KO) and age-matched wild-type C57BL/6 J mice (WT) received weekly vehicle PBS injections for 12 weeks, while a third group of wild-type mice were injected weekly for 12 weeks with 30 mg/kg body weight humanized GIP-mAb (AB) to assess the possibility of long-term effects of peripheral GIP antagonism on rodent memory and behavior. All mice groups then underwent a battery of tests that evaluated motor behavior, body coordination, and memory. Performance deficits in several memory studies after 12 weeks of treatment were demonstrated in KO, but not in AB or WT mice. Body coordination performance showed no significant differences among the 3 groups. A similar short-term study (3 injections over 9 days) was also conducted and the results were similar to those from the long-term study. Thus, short-term and long-term peripheral GIP antagonism by GIP-mAb did not appear to affect learning and memory in mice, consistent with the notion that the GIP-mAb does not cross the blood brain barrier. Furthermore, our studies indicate that GIP signaling in the brain appears to involve local neurocrine pathways.

Optimal Omeprazole Dosing and Symptom Control: A Randomized Controlled Trial (OSCAR Trial).

BACKGROUND: Proton pump inhibitors (PPIs) are potent inhibitors of acid secretion and are the mainstay of therapy for gastroesophageal reflux disease (GERD). Initially designed to be taken 30 min before the first daily meal, these agents are commonly used suboptimally, which adversely affects symptom relief. No study to date has assessed whether correcting dosing regimens would improve symptom control. The objective of this study was to determine whether patients with persistent GERD symptoms on suboptimal omeprazole dosing experience symptomatic improvement when randomized to commonly recommended dosing regimen and to evaluate the economic impact of suboptimal PPI dosing in GERD patients. METHODS: Patients with persistent heartburn symptoms ≥ 3 times per week treated with omeprazole 20 mg daily were enrolled and randomized to commonly recommended dosing or continued suboptimal dosing of omeprazole. The primary outcomes were changes in symptom, frequency, and severity, as determined using the Gastroesophageal Reflux Disease Symptom Assessment Scale (GSAS) 4 weeks after the intervention was administered. In secondary analysis, an alternative measure of symptom load was used to infer potential costs. RESULTS: Sixty-four patients were enrolled. GSAS symptom, frequency, and severity scores were significantly better when dosing was optimized for overall and heartburn-specific symptoms (P < 0.01 for all parameters). Cost savings resulting from reduced medical care and workplace absenteeism were estimated to be $159.60 per treated patient, with cost savings potentially exceeding $4 billion annually in the USA. DISCUSSION: Low-cost efforts to promote commonly recommended PPI dosing can dramatically reduce GERD symptoms and related economic costs. ClinicalTrials.gov, number: NCT02623816.

Gastric inhibitory polypeptide immunoneutralization attenuates development of obesity in mice.

Previous reports have suggested that the abrogation of gastric inhibitory polypeptide (GIP) signaling could be exploited to prevent and treat obesity and obesity-related disorders in humans. This study was designed to determine whether immunoneutralization of GIP, using a newly developed specific monoclonal antibody (mAb), would prevent the development of obesity. Specific mAb directed against the carboxy terminus of mouse GIP was identified, and its effects on the insulin response to oral and to intraperitoneal (ip) glucose and on weight gain were evaluated. Administration of mAb (30 mg/kg body wt, BW) to mice attenuated the insulin response to oral glucose by 70% and completely eliminated the response to ip glucose coadministered with human GIP. Nine-week-old C57BL/6 mice injected with GIP mAbs (60 mg·kg BW(-1)·wk(-1)) for 17 wk gained 46.5% less weight than control mice fed an identical high-fat diet (P < 0.001). No significant differences in the quantity of food consumed were detected between the two treatment groups. Furthermore, magnetic resonance imaging demonstrated that subcutaneous, omental, and hepatic fat were 1.97-, 3.46-, and 2.15-fold, respectively, lower in mAb-treated animals than in controls. Moreover, serum insulin, leptin, total cholesterol (TC), low-density lipoprotein (LDL), and triglycerides were significantly reduced, whereas the high-density lipoprotein (HDL)/TC ratio was 1.25-fold higher in treated animals than in controls. These studies support the hypothesis that a reduction in GIP signaling using a GIP-neutralizing mAb might provide a useful method for the treatment and prevention of obesity and related disorders.

Consumer use of over-the-counter proton pump inhibitors in patients with gastroesophageal reflux disease.

OBJECTIVES: Optimal administration of proton pump inhibitor (PPI) for the treatment of gastroesophageal reflux disease (GERD) requires consideration of meal timing. Since becoming available over the counter (OTC), no studies have assessed treatment patterns and symptom control in OTC consumers. The objective of this study was to survey dosing patterns and symptom control in OTC and prescription PPI users. METHODS: Patients at five clinics were surveyed regarding diagnosis of GERD, use of OTC or prescription PPIs, information on time of day dosing, demographics, and Gastroesophageal Reflux Disease Symptom Assessment Scale (GSAS; 2001, Johnson & Johnson). RESULTS: Of the 1,959 patients surveyed, 610 (31%) used PPIs for GERD. Of these, 190 (31%) and 223 (37%) received prescriptions from gastroenterologists (GIs) and primary care physicians (PCPs), respectively; 197 (32%) purchased OTC PPIs. Of the patients prescribed PPIs by GIs, 71% were optimal users, whereas 47% of patients receiving prescriptions from PCPs and 39% of consumers used PPIs optimally (P<0.001 compared with GIs). GSAS symptom, frequency, and severity scores were significantly better in patients prescribed PPIs by GIs (all P<0.001, GI compared with PCP and consumer). GSAS symptom, frequency, and severity scores were also significantly better in patients using PPIs optimally (P<0.001 for all parameters) compared with those taking PPIs suboptimally or excessively. CONCLUSIONS: Patients receiving prescription PPI from a GI are more likely to be optimal users with better symptom control. Conversely, consumers are more likely to be suboptimal users with inadequate symptom control.

Obesity and the gastrointestinal tract: you are what you eat.

Obesity represents a complex multifactorial syndrome that develops from interactions among genetic and environmental factors and is a leading cause of illness and death. The prevalence of obesity in the United States has increased dramatically since 1975. Although often ignored, the gastrointestinal tract, and the gastrointestinal regulatory peptides in particular, constitutes an ideal starting point for defining and investigating obesity as it represents the route by which all nutrients are ingested, processed, and absorbed. Another important factor to consider when evaluating the etiology of obesity is the capacity for all animals to store nutrients. Insulin is the most potent anabolic hormone, and it appears to have evolved from the need to maximize energy efficiency, obviating the requirement to continuously forage for food. Organisms expressing this important peptide possessed a distinct survival advantage and flourished. During the course of evolution, insulin biosynthesis translocated from the intestine to pancreatic islets, which necessitated a messenger from the intestine to complete the "enteroinsular axis." The eventual development of glucose-dependent insulinotropic polypeptide (GIP) and other incretins fulfilled this requirement. GIP appears to offer an additional survival benefit by not only stimulating intestinal glucose transport and maximally releasing insulin to facilitate nutrient storage but also by its insulin-mimetic properties, including enhanced uptake of glucose by adipocytes. This physiological redundancy offered by insulin and GIP ensured the survival of organisms during times when food was scarce. As food is no longer scarce, at least in the West, this survival advantage appears to have contributed to the current obesity epidemic.

Evolutionary expression of glucose-dependent-insulinotropic polypeptide (GIP).

Glucose-dependent insulinotropic polypeptide (GIP) is a mammalian incretin hormone released into the circulation following nutrient ingestion. We examined the functional evolution of GIP and its relationship with insulin to delineate their respective roles in promoting nutrient efficiency. Expression patterns were examined in the sea lamprey (Petromyzon marinus), a basal vertebrate lacking a distinct pancreas, and in the zebrafish, Xenopus laevis, chicken, and mouse, organisms possessing extraintestinal pancreata. Although sea lamprey genomic analysis predicted a potential GIP-like gene, transcripts were not detected, and insulin expression was confined to the caudal pancreatic bud. GIP was detected in both the intestine and pancreas of the zebrafish and X. laevis. In contrast, GIP and insulin expression were limited to the intestine and pancreas, respectively, in chicken and mouse. Phylogenetic analysis of the glucagon-like ligands suggested proglucagon as the common ancestor, supporting the theory that GIP arose as a gene duplication of proglucagon. Insulin-secreting cells in the sea lamprey intestine may have obviated the need for an enteroinsular axis, and zebrafish may represent an evolutionary transition where GIP does not yet function as an incretin hormone. These observations are consistent with the hypothesis that GIP and insulin influence survival advantage by enhancing the efficiency of nutrient absorption and energy storage.

Evolutionary conservation of glucose-dependent insulinotropic polypeptide (GIP) gene regulation and the enteroinsular axis.

Glucose-dependent insulinotropic polypeptide (GIP), an important component of the enteroinsular axis, is a potent stimulator of insulin secretion, functioning to maintain nutrient efficiency. Although well-characterized in mammals, little is known regarding GIP transcriptional regulation in Danio rerio (Dr). We previously demonstrated that DrGIP is expressed in the intestine and the pancreas, and we therefore cloned the Dr promoter to compare GIP transcriptional regulation in Dr and mammals. Although no significant homology was indentified between the highly conserved mammalian promoter and the DrGIP promoter, 1072-bp of the DrGIP promoter conferred tissue-specific expression in mammalian cell lines. Deletional analysis of the DrGIP promoter identified two regions that, when deleted, reduced transcription by 75% and 95%, respectively. Mutational analysis of the upstream region suggested involvement of an Nkx binding site, although we were unable to identify the factor binding to this site. The cis element in the downstream region was found to be a GATA binding site. Lastly, overexpression and shRNA experiments identified PAX4 as a potential repressor of DrGIP expression. These findings provide evidence that despite the identification of species-specific transcriptional regulators and differences in GIP expression patterns between D. rerio and mammals, a moderate degree of regulatory conservation appears to exist.

Glucose-dependent insulinotropic polypeptide stimulates the proliferation of colorectal cancer cells.

Although numerous epidemiological studies have provided convincing evidence for an increase in the prevalence of colorectal cancer (CRC) in obese individuals, the precise mechanisms involved have not been elucidated. Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal regulatory peptide whose primary physiologic role is to stimulate postprandial pancreatic insulin secretion. Like insulin, GIP has been linked to enhanced nutrient efficiency, which occurred during the course of evolution. Its expression is increased in obesity, and we thus initiated studies to examine whether GIP might contribute to the pathogenesis of obesity-related CRC. RT-PCR and Western analysis demonstrated the presence of the GIP receptor (GIPR) in several human CRC cell lines. GIP stimulated the proliferation of MC-26 cells, a mouse CRC cell line, in a concentration-dependent manner. Western analysis showed that GIP induced the activity of several downstream signaling molecules known to be involved in cellular proliferation in a concentration- and time-dependent manner. These studies indicate that the presence of GIP receptors in CRC may enable ligand binding and, in so doing, stimulate CRC cell proliferation. The overexpression of GIP, which occurs in obesity, might thereby be contributing to the enhanced rate of carcinogenesis observed in obesity.

Expression of glucose-dependent insulinotropic polypeptide in the zebrafish.

In mammals, glucose-dependent insulinotropic polypeptide (GIP) is synthesized predominately in the small intestine and functions in conjunction with insulin to promote nutrient deposition. However, little is known regarding GIP expression and function in early vertebrates like the zebrafish, a model organism representing an early stage in the evolutionary development of the compound vertebrate pancreas. Analysis of GIP and insulin (insa) expression in zebrafish larvae by RT-PCR demonstrated that although insa was detected as early as 24 h postfertilization (hpf), GIP expression was not demonstrated until 72 hpf, shortly after the completion of endocrine pancreatic development but prior to the commencement of independent feeding. Furthermore, whole mount in situ hybridization of zebrafish larvae showed expression of GIP and insa in the same tissues, and in adult zebrafish, RT-PCR and immunohistochemistry demonstrated GIP expression in both the intestine and the pancreas. Receptor activation studies showed that zebrafish GIP was capable of activating the rat GIP receptor. Although previous studies have identified four receptors with glucagon receptor-like sequences in the zebrafish, one of which possesses the capacity to bind GIP, a functional analysis of these receptors has not been performed. This study demonstrates interactions between the latter receptor and zebrafish GIP, identifying it as a potential in vivo target for the ligand. Finally, food deprivation studies in larvae demonstrated an increase in GIP and proglucagon II mRNA levels in response to fasting. In conclusion, the results of these studies suggest that although the zebrafish appears to be a model of an early stage of evolutionary development of GIP expression, the peptide may not possess incretin properties in this species.

False-positive secretin stimulation test for gastrinoma associated with the use of proton pump inhibitor therapy.

We report the case of a 22-year-old woman who was referred for evaluation of possible Zollinger-Ellison syndrome because of hypergastrinemia and a positive secretin stimulation test. She was being treated with proton pump inhibitors (PPIs) for severe gastroesophageal reflux disease during her initial evaluation. At cessation of PPI therapy, her fasting serum gastrin levels normalized, as did her response to secretin injection. Previous reports describing false-positive secretin tests have been limited to cases of hypergastrinemia in the setting of chronic atrophic gastritis, presumably a result of achlorhydria. This case represents a clearly documented instance of PPI-related hypergastrinemia with a false-positive secretin test, with subsequent normalization of serum gastrin and a negative secretin test after withdrawal of PPI therapy. The current case emphasizes the need to assess the acid secretory status of individuals with hypergastrinemia and to discontinue the use of potent antisecretory agents, principally PPIs, to avoid the erroneous diagnosis of gastrinoma and before embarking on expensive and potentially invasive evaluations for the purpose of tumor localization.

Glucose-dependent insulinotropic polypeptide enhances adipocyte development and glucose uptake in part through Akt activation.

BACKGROUND & AIMS: In addition to its role as the primary mediator of the enteroinsular axis, glucose-dependent insulinotropic polypeptide (GIP) may play a critical role in the development of obesity. The purpose of these studies was to characterize the effects of GIP and its receptor (GIPR) in adipocyte development and signaling. METHODS: Effects of GIP and GIPR on differentiated 3T3-L1 cells were analyzed using Western blot analysis, Oil-Red-O staining, cyclic adenosine monophosphate radioimmunoassay, immunofluorescence microscopy, and glucose uptake measurements. RESULTS: To determine whether GIP and GIPR are important components in adipocyte development, the expression profile of GIPR during differentiation was examined. GIPR protein expression was enhanced during the differentiation process, and coincubation with its ligand GIP augmented the expression of aP2, a fat cell marker. Conversely, the suppression of GIPR expression by a specific short hairpin RNA attenuated Oil-Red-O staining and aP2 expression, suggesting that the GIPR may play a critical role in adipocyte development. To investigate specific signaling components that may mediate the effects of GIP, we analyzed Akt, glucose transporter-4, and glucose uptake, all of which are modulated by insulin in fat cells. Like insulin, GIP induced the activation of Akt in a concentration-dependent manner, promoted membrane glucose transporter-4 accumulation, and enhanced [(3)H]-2-deoxyglucose uptake. CONCLUSIONS: These studies provide further evidence for an important physiologic role for GIP in lipid homeostasis and possibly in the pathogenesis of obesity. Furthermore, our data indicate that the GIPR might represent a suitable target for the treatment of obesity.

Gastrointestinal regulatory peptides: an overview.

PURPOSE OF REVIEW: This review is intended to provide an overview of this section on 'Gastrointestinal regulatory peptides' and to emphasize both similarities and differences between 'classic' hormones and those peptides synthesized within, and released from, the gastrointestinal tract. It will also discuss recent investigation involving these peptides and their physiological properties and pathologic potential. RECENT FINDINGS: More recent investigation, much of which is discussed in this section, has looked at the central role of the gastrointestinal tract, and specifically gastrointestinal regulatory peptides, in nutrient homeostasis and in the pathogenesis of obesity and other nutritional disorders. SUMMARY: Regulatory peptides are chemical messengers that provide a means of communication between two cells which are commonly located in different organ systems. The peptides interact via a shared aqueous environment; whereas this environment is endocrine in nature for classic hormones and gastrointestinal peptides, the latter also include those peptides that communicate more directly with their target via paracrine, neurocrine, and autocrine routes. The field of gastrointestinal regulatory peptides is in its infancy, and the coming decades will witness the development of these peptides, as well as analogues and antagonists, as potential new forms of therapy of obesity and other nutrition-related disorders, as well as other maladies.

Glucose-dependent insulinotropic polypeptide and its role in obesity.

PURPOSE OF REVIEW: As we strive to improve our understanding of the factors that contribute to the pathogenesis of obesity, it is certainly logical to speculate that a nutrition-dependent component emanating from the gastrointestinal tract, which has the capacity to regulate insulin expression, could represent an etiologic factor in this serious health issue. One such regulatory peptide that possesses these properties is glucose-dependent insulinotropic polypeptide. Release of this peptide is stimulated by the ingestion of glucose and fat from the upper portion of the small intestine, which in turn enhances the release of insulin from the pancreas. RECENT FINDINGS: In addition to its indirect effects in inducing obesity through the stimulation of insulin, a potent efficiency hormone, glucose-dependent insulinotropic polypeptide, like insulin, also directly modulates a variety of factors that are important in adipocyte homeostasis. Such factors include lipoprotein lipase, Akt, and GLUT-4, all of which promote the storage of fat. SUMMARY: In this review, various mechanisms by which glucose-dependent insulinotropic polypeptide may serve as a link that indirectly and directly contributes to the development of obesity will be discussed. Understanding the peptide's role will potentially help provide novel ways to prevent and treat obesity.

Glucose-dependent insulinotropic polypeptide modulates adipocyte lipolysis and reesterification.

OBJECTIVE: Glucose-dependent insulinotropic polypeptide (GIP) is an incretin released from intestinal K-cells during the postprandial period. Previous studies have suggested that GIP may play an etiologic role in obesity; thus, the GIP receptor may represent a target for anti-obesity drugs. The present studies were conducted to elucidate mechanisms by which GIP might promote obesity by examining the effect of GIP on both glycerol release (indicative of lipolysis) and free fatty acid (FFA) release (indicative of both lipolysis and reesterification), as well as the ability of a GIP-specific receptor antagonist (ANTGIP) to attenuate these effects. RESEARCH METHODS AND PROCEDURES: Isolated rat adipocytes were perifused on a column with 10 nM GIP alone or in combination with 10 microU/mL insulin, 1 microM isoproterenol, or 1 microM ANTGIP. Samples were collected every minute and assayed for FFA, glycerol, and lactate. RESULTS: GIP significantly increased FFA reesterification (decreased FFA release by 25%), stimulated lipolysis (increased glycerol release by 22%), and attenuated the lipolytic response to isoproterenol by 43%. These properties were similar to those of insulin in vitro, suggesting that GIP possesses insulin-like lipogenic effects on adipocytes. Finally, ANTGIP reversed the effects of GIP on both basal and stimulated adipocyte metabolism. DISCUSSION: These studies provide further evidence for an important physiological role for GIP in lipid homeostasis and possibly in the pathogenesis of obesity. They also suggest that the GIP receptor may represent an excellent target for the prevention and treatment of obesity and obesity-related type 2 diabetes.

Attenuation of peroxisome proliferator-activated receptor gamma (PPARgamma) mediates gastrin-stimulated colorectal cancer cell proliferation.

Peroxisome proliferators-activated receptor gamma (PPARgamma) has been shown to suppress cell proliferation and tumorigenesis, whereas the gastrointestinal regulatory peptide gastrin stimulates the growth of neoplastic cells. The present studies were directed to determine whether changes in PPARgamma expression might mediate the effects of gastrin on the proliferation of colorectal cancer (CRC). Initially, using growth assays, we determined that the human CRC cell line DLD-1 expressed both functional PPARgamma and gastrin receptors. Amidated gastrin (G-17) attenuated the growth suppressing effects of PPARgamma by decreasing PPARgamma activity and total protein expression, in part through an increase in the rate of proteasomal degradation. G-17-induced degradation of PPARgamma appeared to be mediated through phosphorylation of PPARgamma at serine 84 by a process involving the biphasic phosphorylation of ERK1/2 and activation of the epidermal growth factor receptor (EGFR). These results were confirmed through the use of EGFR antagonist AG1478 and MEK1 inhibitor PD98059. Furthermore, mutation of PPARgamma at serine 84 reduced the effects of G-17, as evident by inability of G-17 to attenuate PPARgamma promoter activity, degrade PPARgamma, or inhibit the growth suppressing effects of PPARgamma. The results of these studies demonstrate that the trophic properties of gastrin in CRC may be mediated in part by transactivation of the EGFR and phosphorylation of ERK1/2, leading to degradation of PPARgamma protein and a decrease in PPARgamma activation.

Sp1/Sp3 binding is associated with cell-specific expression of the glucose-dependent insulinotropic polypeptide receptor gene.

The physiological effects of glucose-dependent insulinotropic polypeptide (GIP) are mediated through specific receptors expressed on target cells. Because aberrant GIP receptor (GIPR) expression has been implicated in abnormal GIP responses associated with type 2 diabetes mellitus and food-induced Cushing's syndrome, we sought to identify factors that regulate the GIPR. We previously demonstrated that sequences between -1 and -100 of the GIPR gene were sufficient to direct transcription in a rat insulinoma cell line (RIN38). In the present study, we compared the 5'-flanking regions of the rat and human GIPR gene and demonstrated 88% identity within the first 92 bp. Subsequent serial deletion analyses showed that the region between -85 and -40 is essential for maximal promoter activity. Within this region, we identified three putative Sp1 binding motifs, located at positions -77, -60, and -50, that can specifically bind both Sp1 and Sp3. Whereas mutation of the Sp1 sites at -50 and -60 led to 36 and 40% reduction in promoter activity, respectively, mutation of the Sp1 motif at -70 did not affect promoter activity. Cotransfection of S2 Schneider cells with GIPR-luciferase chimeric constructs and either Sp1 or Sp3 expression vectors indicated that both Sp1 and the long form of Sp3 activate transcription through binding to the Sp1 sites located between -100 and -40. Lastly, chromatin immunoprecipitation analyses revealed that both Sp1 and Sp3 bind to the GIPR promoter region in RIN38 cells. These results indicate that cell-specific expression of GIPR is associated with the binding of the transcription factors Sp1 and Sp3 to the GIPR promoter.

Vitamin D deficiency enhances the growth of MC-26 colon cancer xenografts in Balb/c mice.

Vitamin D deficiency has been associated with increased risk of colon cancer in epidemiologic and prospective clinical studies. In vitro and in vivo studies demonstrated that 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and its analogs inhibit colon cancer cell proliferation. Few studies have evaluated the effect of vitamin D deficiency on the development and growth of colon cancer. To assess the antiproliferative effects of 25-hydroxyvitamin D [25(OH)D] and 1,25(OH)2D3 in vitro, we cultured MC-26 (a colon cancer cell line) in the presence of 25(OH)D3 and 1,25(OH)2D3 and performed [3H]thymidine incorporation. The proliferation of MC-26 was significantly inhibited by both 25(OH)D3 and 1,25(OH)2D3. To determine the effect of vitamin D deficiency on colon cancer proliferation, Balb/c mice were rendered vitamin D deficient by feeding them a vitamin D-deficient diet for 3 mo. A group of vitamin D-sufficient mice was given the same diet with supplemental vitamin D. The mice were injected with MC-26 colon cancer cells and the tumors were measured daily for 20 d. Vitamin D-sufficient mice had 40% smaller tumors than vitamin D-deficient mice. The tumors were evaluated for mRNA expression of the vitamin D receptor (VDR) and 25-hydroxvitamin D-1alpha-hydroxylase (1alpha-OHase) by quantitative RT-PCR. The expression of the mRNA for the VDR and the 1alpha-OHase was 37- and 6-fold higher, respectively, in the vitamin D-sufficient mice compared with the vitamin D-deficient mice. We conclude that vitamin D deficiency enhances the growth of colon cancer in mice. The tumor expression of VDR and 1alpha-OHase indicates possible autocrine/paracrine cell growth regulation by vitamin D.

Peroxisome proliferator-activated receptor gamma inhibition prevents adhesion to the extracellular matrix and induces anoikis in hepatocellular carcinoma cells.

Activation of the nuclear transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits growth and survival of hepatocellular carcinoma (HCC) cell lines. To further investigate the function of PPARgamma in HCC, PPARgamma expression patterns in primary tumors were examined, and the responses of two HCC cell lines to PPARgamma activation and inhibition were compared. PPARgamma expression was increased in HCC and benign-appearing peritumoral hepatocytes compared with remote benign hepatocytes. Both compound PPARgamma inhibitors and PPARgamma small interfering RNAs prevented HCC cell lines from adhering to the extracellular matrix. Loss of adhesion was followed by caspase-dependent apoptosis (anoikis). PPARgamma inhibitors had no effect on initial beta1 integrin-mediated adhesion, or on total focal adhesion kinase levels but did reduce focal adhesion kinase phosphorylation. The PPARgamma inhibitor T0070907 was significantly more efficient at causing cancer cell death than the activators troglitazone and rosiglitazone. T0070907 caused cell death by reducing adhesion and inducing anoikis, whereas the activators had no direct effect on adhesion and caused cell death at much higher concentrations. In conclusion, PPARgamma overexpression is present in HCC. Inhibition of PPARgamma function causes HCC cell death by preventing adhesion and inducing anoikis-mediated apoptosis. PPARgamma inhibitors represent a potential novel treatment approach to HCC.

Effects of nonselective cyclooxygenase inhibition with low-dose ibuprofen on tumor growth, angiogenesis, metastasis, and survival in a mouse model of colorectal cancer.

PURPOSE: To determine whether the nonselective and relatively inexpensive nonsteroidal anti-inflammatory drug ibuprofen would be effective in inhibiting colorectal cancer and might improve mortality in a mouse model. EXPERIMENTAL DESIGN: The effects of ibuprofen on tumor growth inhibition and animal survival have been examined in both mouse and human colorectal cancer tumor models. Angiogenesis was measured by in vitro endothelial cell tube formation and immunohistochemistry. RESULTS: Ibuprofen significantly inhibited cell proliferation in mouse (MC-26) and human (HT-29) colorectal cancer cell lines. In vitro angiogenesis assays also indicated that ibuprofen decreased both cell proliferation and tube formation. The administration of chow containing 1,360 ppm ibuprofen, which achieved an average plasma concentration of ibuprofen lower than the peak level achieved in humans at therapeutic doses, inhibited tumor growth by 40% to 82%. Fewer liver metastases were found in the ibuprofen group compared with the control group. In combination therapy with the standard antineoplastic agents, 5-fluorouracil, or irinotecan (CPT-11), tumor volumes in the groups with ibuprofen +/- CPT-11 or 5-fluorouracil were smaller than in the control group. Ibuprofen was similar to the cyclooxygenase-2 selective inhibitor rofecoxib in its ability to suppress tumor growth and improve overall survival. CONCLUSIONS: Ibuprofen, in part by modulating tumor angiogenesis, decreases both tumor growth and metastatic potential in mice. The ibuprofen doses were in the low range of therapeutic human plasma concentrations. Ibuprofen potentiates the antitumor properties of CPT-11 and improves survival of mice without increasing gastrointestinal toxicity.