Frondoside A enhances the antiproliferative effects of gemcitabine in pancreatic cancer.

Pancreatic cancer has a very poor prognosis. While gemcitabine is the mainstay of therapy and improves quality of life, it has little impact on survival. More effective treatments are desperately needed for this disease. Frondoside A is a triterpenoid glycoside isolated from the Atlantic sea cucumber, Cucumaria frondosa. Frondoside A potently inhibits pancreatic cancer cell growth and induces apoptosis in vitro and in vivo. The aim of the present study was to investigate whether frondoside A could enhance the anti-cancer effects of gemcitabine. Effects of frondoside A and gemcitabine alone and in combination on proliferation were investigated in two human pancreatic cancer cell lines, AsPC-1 and S2013. To investigate possible synergistic effects, combinations of low concentrations of the two drugs were used for a 72 h treatment period in vitro. Growth inhibition was significantly greater with the drug combinations than their additive effects. Combinations of frondoside A and gemcitabine were tested in vivo using the athymic mouse model. Xenografts of AsPC-1 and S2013 cells were allowed to form tumours prior to treatment with the drugs alone or in combination for 30 days. Tumours grew rapidly in placebo-treated animals. Tumour growth was significantly reduced in all treatment groups. At the lowest dose tested, gemcitabine (4 mg/kg/dose), combined with frondoside A (100 µg/kg/day) was significantly more effective than with either drug alone. To conclude: The present data suggest that combinations of frondoside A and gemcitabine may provide clinical benefit for patients with pancreatic cancer.

Induction of the adenoma-carcinoma progression and Cdc25A-B phosphatases by the trefoil factor TFF1 in human colon epithelial cells.

TFF1 is overexpressed in inflammatory diseases and human cancers of the digestive and urogenital systems. To examine the transforming potential of TFF1 in human colon epithelial cells, premalignant PC/AA/C1 adenoma cells (PC) derived from a patient with familial adenomatous polyposis (FAP) were transformed by the TFF1 cDNA and used as a model of the adenoma-carcinoma transition. Constitutive expression of TFF1 increased anchorage-independent cell growth in soft agar, and induced or potentiated the growth of colon PC-TFF1 and kidney MDCKts.src-TFF1 tumor xenografts in athymic mice. This resulted in reduction of thapsigargin-induced apoptosis and promotion of collagen type I invasion through several oncogenic pathways. Using the differential display approach to identify TFF1 target genes, we found that the dual specific phosphatases Cdc25A and B implicated in cell cycle transitions are strongly upregulated under active forms in both PC-TFF1 and HCT8/S11-TFF1 colon cancer cells. Accordingly, TFF1 expression is absent in normal human colon crypts but is induced in correlation with Cdc25a and b transcript levels and tumor grade in familial and sporadic colon adenomas and carcinomas. We propose that TFF1 and Cdc25A-B cooperate with other dominant oncogenic pathways to induce the adenoma and adenocarcinoma transitions. Agents that target TFF1/Cdc25 signaling pathways may be useful for treating patients with TFF1-positive solid tumors.

Neuroprotective effects of leptin in vivo and in vitro.

The present study explored the efficacy of leptin in protecting against in vivo induction of excitotoxic lesions by the glutamatergic analogue ibotenate injected into the developing mouse brain and against in vitro NMDA-induced cell death in primary neuronal cultures. Ibotenate injected intracerebrally (i.c.) to mice on postnatal day 5 produced transcortical necrosis and white matter cysts. Co-treatment with leptin administered i.c. or i.p. reduced ibotenate-induced cortical lesions and white matter cysts by 50%. in vitro, leptin afforded significant neuroprotection of mouse cortical neurons against NMDA cytotoxicity. The neuroprotective effect of leptin was antagonized both in vivo and in vitro by the Jak2 inhibitor AG490, indicating that it was mediated via the leptin receptor and Jak2 activation. These findings are the first evidence for a role of leptin in neuroprotection.

Antral mucosa expresses functional leptin receptors coupled to STAT-3 signaling, which is involved in the control of gastric secretions in the rat.

BACKGROUND & AIMS: Leptin is a circulating hormone that communicates the peripheral nutritional status to the hypothalamus, which controls food intake, energy expenditure, and body weight. This study characterizes leptin receptors and leptin-sensitive STAT proteins in the antrum and investigates the effects of leptin on gastric secretions. METHODS: The effects of leptin on gastrin messenger RNA (mRNA), plasma gastrin, gastric acid in vivo in the rat, and on somatostatin and gastrin secretions by isolated antral cells were determined in vitro. Leptin receptors were investigated in isolated rat antral cells by reverse transcription-polymerase chain reaction and binding of [(125)I]-leptin studies. The effects of in vivo and in vitro leptin on transduction signal STAT proteins were investigated by immunoblotting antral extracts. RESULTS: Peripheral injection of leptin inhibited in a dose-dependent manner, basal gastric secretion, gastrinemia, and mucosal gastrin mRNA in vivo. mRNAs encoding the long (Ob-Rb) and short (Ob-Ra) receptor forms were detected in rat antral mucosa, as were STAT-1, -3, and -5b immunoreactive proteins. Isolated antral cells specifically bound [(125)I]-leptin, and addition of leptin to these cells inhibited the release of somatostatin and increased the release of gastrin. These effects were associated with an increase in nuclear STAT-3 proteins in vitro and in vivo. CONCLUSIONS: This study provides the first molecular evidence for the coexpression of leptin receptors and STAT-3 in antral mucosa. It provides further evidence for the involvement of leptin in the control of gastric secretions.

Proinflammatory role of leptin in experimental colitis in rats benefit of cholecystokinin-B antagonist and beta3-agonist.

Leptin, a hormone primarily secreted from adipocytes, plays a key role in controlling body weight homeostasis. In vitro studies indicate that it is also implicated in immune responses. Hyperleptinaemia has been reported in acute inflammation, especially during the early stages of intestinal inflammation in rats. The present study investigated the possible role of leptin in the pathogenesis of trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats. Since no specific antagonist of leptin is available, a CCK-B antagonist (YM022) and a beta3 agonist (BRL37344) were used in this study to inhibit leptin secretion. Colitis was induced by intracolonic instillation of TNBS in rats. Five TNBS-groups were subcutaneously implanted with micropumps containing: placebo, YM022, BRL37344, BRL37344 and exogenous leptin simultaneously, or leptin alone. At sacrifices, colitis severity was assessed by macroscopic and histological scoring systems and by determination of tissue myeloperoxidase activity. The TNBS-induced hyperleptinaemia was significantly reduced by YM022 and BRL37344 (p<0.05). Inhibition of leptin secretion markedly reduced colonic inflammation, whatever the criteria considered (i.e. macroscopic, histological or biochemical). In contrast, administration of exogenous leptin completely abolished the beneficial effect of leptin-lowering drugs on colitis severity. These results provide the first direct evidence for an important deleterious role of leptin in the pathogenesis of experimental intestinal inflammation and suggest that a pro-inflammatory activity is attributable to leptin in vivo. Further studies are required to determine if these results have clinical significance.

The H3 receptor is involved in cholecystokinin inhibition of food intake in rats.

We investigated the peripheral effects of an H3-receptor agonist and an H3-receptor antagonist (R)alpha-methylhistamine (Ralpha-MeHA) and thioperamide, respectively, on basal feeding and the CCK8-induced inhibition of food intake in rat. Intraperitoneal injection of thioperamide reduced food intake in a dose-dependent manner with maximal inhibition (35%, P<0.01 vs saline) at 3 mg/kg. (R)alpha-MeHA (0.3-3 mg/kg i.p.), an H3-receptor agonist alone had no effect on feeding but reversed the thioperamide-induced inhibition of food intake in a dose-dependent manner. The maximal feeding inhibitory dose of thioperamide (3 mg.kg i.p) increased by 40% and 22 % (P<0.01 vs saline) brain and stomach histamine contents, respectively. Histamine (0.3 - 6 mg/kg i.p.) and CCK-8 (3 - 30 microg/kg i.p) also inhibited food intake in a dose-dependent manner. Inhibition was 20% to 40% for histamine and 40% to 80% (P<0.01 vs saline) for CCK8. CCK-8 inhibition of feeding was increased by thioperamide and prevented by (R)alpha-MeHA in a dose-dependent way. In addition, CCK-8 did not reduce food intake if rats were pretreated with pyrilamine or ranitidine postsynaptic H1- and H2-receptor antagonists respectively. Our data suggest that the H3-receptor is involved in basal feeding. They also suggest that CCK satiety depends upon the release of histamine which acts on the H2- and H1-receptors, the final mediators of this effect.

Leptin promotes invasiveness of kidney and colonic epithelial cells via phosphoinositide 3-kinase-, rho-, and rac-dependent signaling pathways.

Leptin plays a key role regulating food intake, body weight and fat mass. These critical parameters are associated with an increased risk for digestive and mammary gland cancer in the Western population. Here we determined whether leptin contributes to the invasive phenotype of colonic and kidney epithelial cells at various stages of the neoplastic progression. First, leptin potently (EC50 = 10-30 ng/ml) induces invasion of collagen gels by premalignant familial adenomatous colonic cells PC/AA/C1 and nontumorigenic MDCK kidney epithelial cells, their src-transformed counterparts, and the human adenocarcinoma colonic cells LoVo and HCT-8/S11. Leptin and its Ob-Rb receptors were consistently identified by RT-PCR and immunoblotting in these cell lines, as well as in human colonic epithelial crypts, polyps, colonic tumor resections, and adjacent mucosa. Leptin-induced invasion was effectively blocked by pharmacological inhibitors of several downstream signaling pathways involved in cell transformation, namely, JAK2 tyrosine kinase (AG490), phosphoinositide PI3'-kinase (wortmannin and LY294002), mTOR kinase (rapamycin), and protein kinases C (GF109203X, Gö6976). Accordingly, leptin induces transient elevation of the PI3'-kinase lipid products in JAK2 immunoprecipitates prepared from parental MDCK cells. The leptin effect on invasion was potentiated by the activated form of the small GTPase RhoA and was abrogated by dominant negative mutants of RhoA, Rac1, and the p110alpha of PI3'-K. Our data indicate that leptin may exert a local and beneficial effect on migration of normal colonic epithelial cells and reparation of the inflamed or wounded digestive mucosa. We also emphasize a new role for leptin, linking the nutritional and body fat status to digestive cancer susceptibility by stimulating the invasive capacity of colonic epithelial cells at early stages of neoplasia. This finding has potential clinical implications for colon cancer progression and management of obesity.

Leptin secretion and leptin receptor in the human stomach.

BACKGROUND AND AIM: The circulating peptide leptin produced by fat cells acts on central receptors to control food intake and body weight homeostasis. Contrary to initial reports, leptin expression has also been detected in the human placenta, muscles, and recently, in rat gastric chief cells. Here we investigate the possible presence of leptin and leptin receptor in the human stomach. METHODS: Leptin and leptin receptor expression were assessed by immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR), and western blot analysis on biopsy samples from 24 normal individuals. Fourteen (10 healthy volunteers and four patients with non-ulcer dyspepsia and normal gastric mucosa histology) were analysed for gastric secretions. Plasma and fundic mucosa leptin content was determined by radioimmunoassay. RESULTS: In fundic biopsies from normal individuals, immunoreactive leptin cells were found in the lower half of the fundic glands. mRNA encoding ob protein was detected in the corpus of the human stomach. The amount of fundic leptin was 10.4 (3.7) ng leptin/g mucosa, as determined by radioimmunoassay. Intravenous infusions of pentagastrin or secretin caused an increase in circulating leptin levels and leptin release into the gastric juice. The leptin receptor was present in the basolateral membranes of fundic and antral gastric cells. mRNA encoding Ob-RL was detected in both the corpus and antrum, consistent with a protein of approximately 120 kDa detected by immunoblotting. CONCLUSION: These data provide the first evidence of the presence of leptin and leptin receptor proteins in the human stomach and suggest that gastric epithelial cells may be direct targets for leptin. Therefore, we conclude that leptin may have a physiological role in the human stomach, although much work is required to establish this.

Physiological role of cholecystokinin B/gastrin receptor in leptin secretion.

In the present study, we investigated whether cholecystokinin (CCK) or its structurally related peptide gastrin participates in long term regulation of adipocyte leptin secretion. The levels of circulating leptin observed after 2 and 6 h of refeeding in 18-h fast rats were significantly lowered by injection of the specific gastrin/CCK-B receptor antagonist YM022 at doses that did not affect feeding behavior. Moreover, in normally fed animals, circulating leptin was markedly decreased by chronic injection of YM022 (from 4 +/- 0.6 to 2.1 +/- 0.5 ng/ml). Consistent with these observations, YM022 treatment decreased leptin messenger RNA (mRNA) levels and increased the leptin content in rat epididymal fat tissue. Rat adipocytes exclusively contain gastrin/CCK-B receptor mRNA, but not CCK-A receptor mRNA. Furthermore, adipocyte membranes bound [125I]CCK-8 in a saturable manner, with kinetics consistent with a single class of high affinity sites with a Kd of 0.2 nM. These data argue for a physiological role for the CCK-B/gastrin receptor in adipocyte leptin regulation. We therefore propose that gastrin is involved in long term regulation of leptin expression and secretion in rat fat tissues through activation of an adipocyte gastrin/CCK-B receptor.

[Gastrin-17 and G17-gly induce proliferation of LoVo cells through the CCK B/gastrin receptor]. / La G17 et la G17-gly stimulent la prolifération des cellules LoVo par l'intermédiaire du récepteur CCK B/gastrine.

BACKGROUND AND METHODS: Recent studies suggest that glycine-extended gastrin (G17-gly) stimulates in vitro proliferation of the pancreatic cell line AR4-2J, through selective receptors distinct from the CCK-B/G-receptor mediating the effects of amidated gastrin (G17). The aims of our study were to examine the effects of G17 and G17-gly on the growth of the colorectal cancer cell line LoVo and to determine the receptor involved by using selective receptor-antagonist. RESULTS: Both G17 and G17-gly stimulated [3H]-thymidine incorporation in a concentration-dependent fashion. Maximal stimulation (153 +/- 18% and 166 +/- 17% of control, p < 0.01) was achieved with 10 nM G17 and 100 nM G17-gly, respectively. These stimulations were fully prevented by the presence of 10 pM YM022, a G/CCK B receptor-antagonist, but unaffected by L364,718, a CCK A receptor-antagonist. Basal growth of LoVo cells was inhibited by YM022 and stimulated by L364,718. CCK A and G/CCK B receptors mRNA were detected in the cells. Gastrin immunoreactivity was detected in the cells (16 pM) and in the extracellular medium (4.5 pM). CONCLUSION: Both G17 and G17-gly stimulate LoVo cells growth through the activation of a gastrin/CCK B receptor. The evidence for secreted gastrin and CCK A and B receptors mRNA may further suggest the existence of an autocrine loop involving a stimulatory gastrin/CCK B receptor.

The stomach is a source of leptin.

The circulating peptide leptin, which is the product of the ob gene, provides feedback information on the size of fat stores to central Ob receptors that control food intake and body-weight homeostasis. Leptin has so far been reported to be secreted only by adipocytes and the placenta. Here we show that leptin messenger RNA and leptin protein are present in rat gastric epithelium, and that cells in the glands of the gastric fundic mucosa are immunoreactive for leptin. The physiological function of this previously unsuspected source of leptin is unknown. However, both feeding and administration of CCK-8 (the biologically active carboxy-terminal end of cholecystokinin) result in a rapid and large decrease in both leptin cell immunoreactivity and the leptin content of the fundic epithelium, with a concomitant increase in the concentration of leptin in the plasma. These results indicate that gastric leptin may be involved in early CCK-mediated effects activated by food intake, possibly including satiety.

YM022, a highly potent and selective CCKB antagonist inhibiting gastric acid secretion in the rat, the cat and isolated rabbit glands.

We investigated the effects of the novel CCKB/gastrin antagonist YM022 on gastric acid secretion in vivo and in vitro, compared to CI-988 and L365,260 as reference antagonists. In the anaesthetized rat, pentagastrin-induced stimulation of gastric acid secretion was dose-dependently and up to 100% inhibited by i.v. administration of YM022 with an ID50 of 0.009 +/- 0.0006 mumol/kg h in comparison to 0.6 +/- 0.03 and 3.40 +/- 0.05 mumol/kg h for CI-988 and L-365,260, respectively. In the gastric fistula cat, i.v. administration of YM022 produced a similar inhibitory effect with an ID50 of 0.02 mumol/kg in comparison to 1.6 and 2.5 mumol/kg for CI-988 and L-365,260, respectively. Furthermore, bolus injection of 0.6 mumol/kg YM022 produced 100% inhibition within 30 min and 85% inhibition was still observed after 3 h. In the isolated rabbit gastric glands, CCK8-stimulated 14C-aminopyrine uptake was inhibited according to the following rank order of potency: YM022 (IC50 = 0.0012 microM) > > CI-988 (IC50 = 0.2 microM) > > L365,260 (IC50 = 2.8 microM). Unlike with L365,260, no influence of CI-988 and YM022 on histamine-stimulated acid output was shown in this study. Thus, YM022 is a highly potent and selective gastric CCKB/gastrin receptor antagonist and has a long-lasting inhibitory effect on gastric acid secretion.

Exposure to ionizing radiation modifies circulating gastrin levels and gastrointestinal endocrine cell densities in the rat.

PURPOSE: Gastrointestinal functions, controlled partly by gut peptides, are disturbed by ionizing radiation exposure. The effect of whole-body irradiation on circulating gastrin levels, densities of gastrointestinal endocrine cells and gastric acid secretion was investigated. MATERIALS AND METHODS: Rats were exposed to 2 or 6 Gy gamma-radiation. They were killed 3 or 7 days later and compared with shams. Plasma gastrin and basal acid output were measured. Endocrine cells were identified by argyrophilia or immunohistochemistry and their densities estimated. RESULTS: Radiation exposure significantly increased gastrinaemia and gastric acid output at the times studied (p<0.05-p<0.001). Endocrine cells displayed different sensitivities to irradiation. In the gastric mucosa, a 6 Gy dose induced a decrease in fundic argyrophil cell, antral gastrin and somatostatin cell densities, always accentuated 7 days after irradiation, while in the intestinal mucosa it induced an increase, with highest values often at 7 days post-irradiation (p<0.01-p<0.001). This was true for neurotensin cells in the jejunum and ileum, substance P cells in ileum and enteroglucagon cells in the descending colon. CONCLUSIONS: Whole-body irradiation in rats significantly alters plasma gastrin levels, and several gut endocrine cell densities. This has repercussions on hormonal function, such as that exerted on acid secretion, and may explain gastrointestinal dysfunction observed following radiation exposure.