Piotr Pierzchalski

Melatonin induces pro-apoptotic signaling pathway in human pancreatic carcinoma cells (PANC-1).

Abstract:  Pancreatic cancer is a highly lethal disease with a poor prognosis for long‐term survival rate at all stages of invasiveness. It responds poorly to radio‐ and chemotherapy because the tumor cells are resistant to apoptosis. Melatonin has been reported to inhibit pancreatic cancer growth in experimental studies in animals but the effect of melatonin on cultured human pancreatic carcinoma cells has not been tested. Moreover, we have recently shown that melatonin stimulates production of two major anti‐apoptotic heat shock proteins, HSP27 and HSP 90, in pancreatic carcinoma cells. This study investigated the changes in intrinsic pathway of apoptosis at the mitochondrial level and cascade of caspases in human pancreatic carcinoma cells (PANC‐1) cells subjected to melatonin and/or luzindole. Melatonin (10−8–10−12 m), the nonselective melatonin receptor antagonist, luzindole (10−8–10−12 m) or a combination of both agents were added to PANC‐1 cell cultures. Cells were harvested, and the cytoplasmic proteins were isolated after 24 and 48 hr of incubation and analyzed employing co‐immunoprecipitation and western blot. Administration of melatonin to the PANC‐1 cells resulted in the stimulation of Bcl‐2/Bax and caspase‐9 proteins levels. The strongest signal of these pro‐apoptotic factors was observed at the low concentration (10−12 m) of melatonin. Pretreatment with luzindole alone and prior to the addition of melatonin reversed the stimulatory effect of this indoloamine on Bcl‐2/Bax and caspase‐9 proteins expression in PANC‐1 cells. This is the first study to demonstrate a pro‐apoptotic effect of low (physiological) concentration of melatonin on the pancreatic carcinoma cells. In conclusion, melatonin induced pro‐apoptotic pathways in human pancreatic carcinoma, probably by interaction with the Mel‐1 A/B receptors.

Leptin in gastroprotection induced by cholecystokinin or by a meal. Role of vagal and sensory nerves and nitric oxide

Leptin, detected recently in the stomach, is a product of the ob gene released by cholecystokinin (CCK) and plays an important role in the control of food intake but its influence on gastroprotection against the damage caused by noxious agents has not been studied. This study was designed to compare the effects of leptin and cholecystokinin-8 (CCK-8) on gastric mucosal lesions induced by topical application of 75% ethanol or acidified aspirin. Four series of Wistar rats (A, B, C and D) were used to determine the effects of: (A) suppression of prostaglandin biosynthesis by indomethacin (5 mg/kg i.p.); (B) inhibition of nitric oxide (NO)-synthase by nitro-L-arginine methyl ester (L-NAME) (5 mg/kg i.v.); (C) blockade of sensory nerves by capsaicin (125 mg/kg s.c.) and (D) bilateral vagotomy, on the gastric lesions induced by intragastric (i.g.) application of ethanol with or without pretreatment with CCK-8, a known gastroprotective substance or leptin. CCK-8 (1-100 microg/kg i.p.) and leptin (0.1-50 microg/kg i.p.) dose dependently attenuated gastric lesions induced by 75% ethanol; the dose reducing these lesions by 50% being about 10 microg/kg and 8 microg/kg, respectively. The protective effects of CCK-8 and leptin were accompanied by a significant rise in gastric blood flow (GBF) and luminal NO concentration. Leptin was also effective to attenuate aspirin-induced damage and the accompanying fall in the GBF, whereas CCK-8 dose dependently worsened aspirin damage and failed to influence GBF. CCK (1-100 microg/kg i.p.), given in graded doses, produced a dose-dependent increase in the plasma leptin level and a rise of the expression of ob messenger RNA (mRNA) in gastric mucosa, the maximum being reached at a dose of 100 microg/kg. Pretreatment with CCK-8 (10 microg/kg i.p.) or with 8% peptone, that is known to stimulate CCK release, also produced a significant rise in plasma leptin levels and up-regulation of ob mRNA while reducing significantly the gastric lesions induced by 75% ethanol to the same extent as that induced by exogenous leptin (10 microg/kg i.p.). Indomethacin, which suppressed prostaglandin generation by approximately 90%, failed to influence leptin- or CCK-8-induced protection against ethanol, whereas L-NAME attenuated significantly CCK-8- and leptin-induced protection and hyperemia but addition to L-NAME of L-arginine, but not D-arginine, restored the protective and hyperemic effects of both hormones. The ob mRNA was detected as a weak signal in the intact gastric mucosa and in that exposed to ethanol alone but this was further enhanced after treatment with graded doses of CCK-8 or peptone meal applied prior to ethanol. We conclude that: (1) exogenous leptin or that released endogenously by CCK or meal exerts a potent gastroprotective action depending upon vagal activity, and involving hyperemia probably mediated by NO and sensory nerves but unrelated to endogenous prostaglandins; (2) leptin mimics the gastroprotective effect of CCK and probably mediates the protective and hyperemic actions of CCK in the rat stomach.

Central Leptin and Cholecystokinin in Gastroprotection against Ethanol-Induced Damage

Background: Leptin, a product of the ob gene controlling food intake, has recently been detected in the stomach and shown to be released by cholecystokinin (CCK) and to induce gastroprotection against various noxious agents, but it is not known whether centrally applied leptin influences gastric secretion and mucosal integrity. Aims: In this study we compared the effects of leptin and CCK-8 applied intracerebroventricularly (i.c.v.) on gastric secretion and gastric mucosal lesions induced by topical application of 75% ethanol. Methods: Several major series of Wistar rats were used in this study. The effects of leptin or CCK applied i.c.v. on gastric secretion were examined using conscious rats with gastric fistulas. For the studies on gastroprotection the following series of rats were used to determine the effects of: (A) leptin and CCK applied centrally on this protection and the blockade of CCKA with loxiglumide (30 mg/kg i.p.) and CCKB receptors with RPR 102681 (30 mg/kg i.p.); (B) cutting of vagal nerves; (C) inactivation of sensory nerves by capsaicin (125 mg/kg s.c.); (D) inhibition of calcitonin gene-related peptide (CGRP) receptors with CGRP8–37 (100 μg/kg i.p.), and (E) suppression of nitric oxide synthase (NOS) with NG-nitro-L-arginine methyl ester (L-NAME) (5 mg/kg i.v.) on ethanol-induced gastric lesions in rats with or without the i.c.v. pretreatment with leptin or CCK-8. Rats were anesthetized 1 h after ethanol administration to measure the gastric blood flow (GBF) and then to determine the area of gastric lesions by planimetry. Blood was withdrawn for the measurement of plasma leptin and gastrin levels by radioimmunoassay and gastric biopsy samples were collected for the determination of cNOS and iNOS mRNA by RT-PCR. Results: Leptin and CCK-8 (0.01–5 μg/kg i.c.v.) dose dependently attenuated gastric lesions induced by 75% ethanol; the doses reducing these lesions by 50% (ED50) were 0.8 and 1.2 μg/kg, respectively. The protective effects of leptin and CCK-8 applied i.c.v. were accompanied by a significant rise in plasma leptin level and an increase in GBF. Blockade of CCKA receptors with loxiglumide abolished the protective and hyperemic effects of CCK but not those of leptin, while RPR 10268, a specific antagonist of CCKB receptors, counteracted leptin-induced protection and the rise in the GBF but failed to influence those afforded by CCK-8. For comparison, pretreatment with peripheral CCK-8 or leptin (10 μg/kg i.p.) causing a similar rise in the plasma leptin level also significantly reduced gastric lesions induced by 75% ethanol. The protective and hyperemic effects of centrally administered leptin were abolished by vagotomy, producing a fall in plasma leptin levels, and significantly attenuated by sensory denervation with capsaicin, by pretreatment with the CGRP antagonist, CGRP8–37, or with L-NAME. A strong signal for iNOS mRNA was recorded in the gastric mucosa of leptin- and CCK-8-treated animals, whereas cNOS mRNA was unaffected. Conclusions: (1) Central leptin exerts a potent gastroprotective action at a dose that has no influence on gastric secretion; (2) this protection depends upon CCKB receptors, vagal activity and sensory nerves, and involves hyperemia probably mediated by NO, and (3) leptin mimics the gastroprotective effect of CCK and may be implicated in the protective and hyperemic actions of this peptide on the rat stomach.

Progastrin and Cyclooxygenase-2 in Colorectal Cancer

Colorectal cancers (CRCs) are one of the most common forms of cancer in Poland and one of the leading causes of death. The tumors have been attributed to genetic, dietary, and other environmental factors, but recently growth factors such as gastrin have also been implicated in the carcinogenesis. The relationship between plasma amidated and nonamidated gastrin in CRCs is controversial. This study was designed (1) to determine the plasma levels of progastrin and amidated gastrin in 50 CRC patients before and 3–6 months after removal of the tumor, (2) to determine the tumor concentrations of these gastrin peptides and the level of expression for gastrin mRNA and gastrin/CCKB receptor mRNA, (3) to examine the expression of cyclooxygenase COX-1 and COX-2 mRNA in CRC tissue, and (4) to compare the prevalence of Hp and its cytotoxic protein, CagA, and cytokines (TNFα, IL-1β, and IL-8) in CRCs, before and after removal of tumor. It was found that the CRC, its resection margin, and the plasma contained severalfold higher levels of progastrin than of amidated gastrins and that the removal of the CRC tumor resulted in a marked reduction in plasma progastrin level without a significant alteration in plasma levels of amidated gastrins. Both gastrin and CCKB-R mRNA were detected in the cancer tissue and resection margin by RT-PCR, and similarly, COX-1 and COX-2 mRNA were expressed in these tissues of most CRCs. The seroprevalence of Hp, especially that expressing CagA, and levels of IL-1β, but not other cytokines, were significantly higher in CRC patients than in 100 age-, gender-, and profession-matched controls and did not change significantly about 3–6 months after tumor resection. We conclude that (1) the CRC and its margin contain large amounts of progastrin and show gene expression of gastrin, CCKB-R, and COX-2; (2) removal of the CRC markedly reduces the plasma concentrations of progastrin; (3) the Hp infection rate is higher in CRC, and this may contribute to colorectal cancerogenesis via enhancement of progastrin and gastrin release; and (4) plasma progastrin concentrations might serve as a biomarker of CRC.

Synthesis of α1-microglobulin in cultured rat hepatocytes is stimulated by interleukin-6, leukemia inhibitory factor, dexamethasone and retinoic acid

The secretion or α1‐microglobulin by primary cultures of rat hepatocytes was found to increase upon the addition of interleukin‐6 or leukemia inhibitory factor, two mediators of acute phase response. This stimulatory effect was further enhanced by dexamethasone. α1‐Microglobulin is synthesized as a precursor also containing bikunin, and the precursor protein is cleaved shortly berore secretion. Our results therefore suggest that both α1‐microglobulin and bikunin are acute phase reactants in rat hepatocytes. Furthermore, we found that retinoic acid, previously shown to be involved in the regulation of cell differentiation and development, also stimulated α1‐microglobulin synthesis. Only free, uncomplexed α1‐microglobulin (28,000 Da) was detected in the hopatocyte media, suggesting that the complex between α1‐microglobulin and α1‐inhibitor 3, found in rat serum, is formed outside the hepatocyte.

Modulation of acute phase protein synthesis in cultured rat hepatocytes by human recombinant hepatocyte growth factor.

Human recombinant hepatocyte growth factor (HGF) added to primary cultures of rat hepatocytes stimulates synthesis of some acute phase proteins, especially alpha-2-macroglobulin. As indicated by changes in mRNA abundance HGF increases alpha-2-macroglobulin production at the pretranslational level. Interleukin-6, the main acute-phase cytokine, does not show synergy with HGF in enhancing synthesis of alpha-2-macroglobulin, and inhibits HGF-induced DNA-synthesis. On the other hand, dexamethasone potentiates the effects of HGF on synthesis of DNA and acute phase proteins by cultured rat hepatocytes.

Effects of Melatonin and Its Analogues on Pancreatic Inflammation, Enzyme Secretion, and Tumorigenesis

Melatonin is an indoleamine produced from the amino acid l-tryptophan, whereas metabolites of melatonin are known as kynuramines. One of the best-known kynuramines is N1-acetyl-N1-formyl-5-methoxykynuramine (AFMK). Melatonin has attracted scientific attention as a potent antioxidant and protector of tissue against oxidative stress. l-Tryptophan and kynuramines share common beneficial features with melatonin. Melatonin was originally discovered as a pineal product, has been detected in the gastrointestinal tract, and its receptors have been identified in the pancreas. The role of melatonin in the pancreatic gland is not explained, however several arguments support the opinion that melatonin is probably implicated in the physiology and pathophysiology of the pancreas. (1) Melatonin stimulates pancreatic enzyme secretion through the activation of entero-pancreatic reflex and cholecystokinin (CCK) release. l-Tryptophan and AFMK are less effective than melatonin in the stimulation of pancreatic exocrine function; (2) Melatonin is a successful pancreatic protector, which prevents the pancreas from developing of acute pancreatitis and reduces pancreatic damage. This effect is related to its direct and indirect antioxidant action, to the strengthening of immune defense, and to the modulation of apoptosis. Like melatonin, its precursor and AFMK are able to mimic its protective effect, and it is commonly accepted that all these substances create an antioxidant cascade to intensify the pancreatic protection and acinar cells viability; (3) In pancreatic cancer cells, melatonin and AFMK activated a signal transduction pathway for apoptosis and stimulated heat shock proteins. The role of melatonin and AFMK in pancreatic tumorigenesis remains to be elucidated.

Long-Lasting Effect of Infant Rats Endotoxemia on Heat Shock Protein 60 in the Pancreatic Acinar Cells: Involvement of Toll-Like Receptor 4

Introduction. Lipopolysaccharide endotoxin (LPS) is responsible for septic shock and multiorgan failure, but pretreatment of rats with low doses of LPS reduced pancreatic acute damage. Aim. We investigated the effects of the endotoxemia induced in the early period of life on Toll-like receptor 4 (TLR4), heat shock protein 60 (HSP60) and proapoptotic Bax, caspase-9 and -3 or antiapoptotic Bcl-2 protein expression in the pancreatic acinar cells of adult animals. Material and Methods. Newborn rats (25 g) were injected with endotoxin (Escherichia coli) for 5 consecutive days. Two months later, pancreatic acinar cells were isolated from all groups of animals and subjected to caerulein stimulation (10−8 M). Protein expression was assessed employing Western blot. For detection of apoptosis we have employed DNA fragmentation ladder assay. Results. Preconditioning of newborn rats with LPS increased TLR4, Caspase-9 and -3 levels, but failed to affect basal expression of HSP60, Bax, and Bcl-2. Subsequent caerulein stimulation increased TLR4, Bcl-2, and caspases, but diminished HSP60 and Bax proteins in pancreatic acinar cells. Endotoxemia dose-dependently increased TLR4, Bax, HSP60, and both caspases protein signals in the pancreatic acini, further inhibiting antiapoptotic Bcl-2. Conclusions. Endotoxemia promoted the induction of HSP60 via TLR4 in the infant rats and participated in the LPS-dependent pancreatic tissue protection against acute damage.

Capsaicin-Sensitive Sensory Nerves Are Necessary for the Protective Effect of Ghrelin in Cerulein-Induced Acute Pancreatitis in Rats

Ghrelin was shown to exhibit protective and therapeutic effect in the gut. Aim of the study was to investigate the role of sensory nerves (SN) in the protective effect of ghrelin in acute pancreatitis (AP). Studies were performed on male Wistar rats or isolated pancreatic acinar cells. After capsaicin deactivation of sensory nerves (CDSN) or treatment with saline, rats were pretreated intraperitoneally with ghrelin or saline. In those rats, AP was induced by cerulein or pancreases were used for isolation of pancreatic acinar cells. Pancreatic acinar cells were incubated in cerulein-free or cerulein containing solution. In rats with intact SN, pretreatment with ghrelin led to a reversal of the cerulein-induced increase in pancreatic weight, plasma activity of lipase and plasma concentration of tumor necrosis factor-α (TNF-α). These effects were associated with an increase in plasma interleukin-4 concentration and reduction in histological signs of pancreatic damage. CDSN tended to increase the severity of AP and abolished the protective effect of ghrelin. Exposure of pancreatic acinar cells to cerulein led to increase in cellular expression of mRNA for TNF-α and cellular synthesis of this cytokine. Pretreatment with ghrelin reduced this alteration, but this effect was only observed in acinar cells obtained from rats with intact SN. Moreover, CDSN inhibited the cerulein- and ghrelin-induced increase in gene expression and synthesis of heat shock protein 70 (HSP70) in those cells. Ghrelin exhibits the protective effect in cerulein-induced AP on the organ and pancreatic acinar cell level. Sensory nerves ablation abolishes this effect.

Effect of Endotoxemia in Suckling Rats on Pancreatic Integrity and Exocrine Function in Adults: A Review Report

Background. Endotoxin (LPS), the component of Gram-negative bacteria, is responsible for sepsis and neonatal mortality, but low concentrations of LPS produced tissue protection in experimental studies. The effects of LPS applied to the suckling rats on the pancreas of adult animals have not been previously explored. We present the impact of neonatal endotoxemia on the pancreatic exocrine function and on the acute pancreatitis which has been investigated in the adult animals. Endotoxemia was induced in suckling rats by intraperitoneal application of LPS from Escherichia coli or Salmonella typhi. In the adult rats, pretreated in the early period of life with LPS, histological manifestations of acute pancreatitis have been reduced. Pancreatic weight and plasma lipase activity were decreased, and SOD concentration was reversed and accompanied by a significant reduction of lipid peroxidation products (MDA + 4 HNE) in the pancreatic tissue. In the pancreatic acini, the significant increases in protein signals for toll-like receptor 4 and for heat shock protein 60 were found. Signal for the CCK1 receptor was reduced and pancreatic secretory responses to caerulein were diminished, whereas basal enzyme secretion was unaffected. These pioneer studies have shown that exposition of suckling rats to endotoxin has an impact on the pancreas in the adult organism.