A novel HIF-1α/VMP1-autophagic pathway induces resistance to photodynamic therapy in colon cancer cells.

Colon cancer is the third most frequent cancer and the fourth most common cause of cancer-related mortality worldwide and the standard therapy is surgical resection plus adjuvant chemotherapy. Photodynamic therapy (PDT) has been proposed as an adjuvant therapy because it can prevent the tumor recurrence after surgical excision in colon cancer patients. Hypoxia is a common feature in solid tumors and leads to chemo/radioresistance. Recently, it has been shown that in response to hypoxia, cells can induce HIF-1α-mediated autophagy to survive in this hostile microenvironment. Moreover, hypoxia and autophagy have been implicated in the resistance to antitumor PDT. However, the molecular signals by which HIF-1α induces autophagy in the PDT context have not been studied yet. Here we evaluate the interplay between HIF-1α and autophagy as well as the underlying mechanism in the PDT resistance of colon cancer cells. Our study demonstrates that HIF-1α stabilization significantly increases VMP1-related autophagy through binding to hypoxia responsive elements in the VMP1 promoter. We show that HIF-1α-induced autophagy increases colon cancer cell survival as well as decreases cell death after PDT. Moreover, here we demonstrate that HIF-1α-induced autophagy is mediated by VMP1 expression, since the downregulation of VMP1 by the RNA interference strategy reduces HIF-1α-induced autophagy and cell survival after PDT. In conclusion, PDT induces autophagy as a survival mechanism and the induction of the novel HIF-1α/VMP1-autophagic pathway may explain, at least in part, the resistance of colon cancer cells to PDT. The knowledge of the molecular mechanisms involved in PDT resistance may lead to more accurate therapeutic strategies.

Nitric oxide and apoptosis induced in Peyer's patches by attenuated strains of Salmonella enterica serovar Enteritidis.

Nitric oxide (NO) is a toxic molecule of the immune system which contributes to the control of microbial pathogens. Additional functions of NO in innate and adaptive immunity have recently been described; these functions include the modulation of the cytokine response of lymphocytes and the regulation of immune cell apoptosis. In addition to direct microbicidal actions, NO has immunoregulatory effects relevant to the control of infections. In turn, infected macrophages and macrophage-regulating lymphocytes may undergo apoptosis during infection by Salmonella spp. In this work we investigated the ability of attenuated strains of Salmonella enterica serovar Enteritidis with different protective capacities to induce intestinal inducible nitric oxide synthase (iNOS) and apoptosis in Peyer's patches (PP) in mice. Results showed that the intestinal iNOS activity correlated with increased apoptosis in PP. Furthermore, the ability to induce intestinal NO production and apoptosis within the first few hours after immunization seemed to correlate with the protective capacity of mutant E/1/3 of S. enterica serovar Enteritidis. It was found that nonprotective mutant C/2/2, which was unable to induce intestinal NO production, also failed to induce apoptosis in PP. Moreover, aminoguanidine treatment at the time of immunization resulted in inhibition of the NO production and apoptosis induced by protective mutant E/1/3 and completely abolished protection against challenge. These results suggest that the induction of iNOS in the intestinal mucosa by attenuated mutant E/1/3 of S. enterica serovar Enteritidis at the time of immunization is necessary to generate a protective immune response.

Molecular and functional characterization of the stress-induced protein (SIP) gene and its two transcripts generated by alternative splicing. SIP induced by stress and promotes cell death.

We have used a quantitative fluorescent cDNA microarray hybridization approach to identify pancreatic genes induced by the cellular stress promoted by acute pancreatitis in the mouse. We report the cloning and characterization of one of them that encodes the stress-induced proteins (SIP). The mouse SIP gene is organized into five exons and expands over approximately 20 kilobase pairs. Exon 4 (38 base pairs) is alternatively spliced to generate two transcripts. Northern blot and in situ hybridization showed that both SIP mRNAs are rapidly and strongly induced in acinar cells of the pancreas with acute pancreatitis. They are also constitutively expressed in several other tissues, although with different ratios. They encode proteins of 18 and 27 kDa (SIP(18) and SIP(27)). SIP(27) is identical to the thymus-expressed acidic protein (TEAP) protein, formerly described as a thymus-specific protein. Expression of the SIP(18) and SIP(27)/EGFP or V5 fusion proteins showed that both are nuclear factors. We monitored SIP expression in NIH3T3 cells submitted to various stress agents. UV stress, base damaging, mutagenic stress, ethanol, heat shock, and oxidative stress induced the concomitant expression of SIP(18) and SIP(27) mRNAs. Finally, transient transfection of SIP(18) and SIP(27) expression plasmids induced death by apoptosis in COS7 cells as measured by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling staining. In conclusion, the SIP gene is an important element of cellular stress response. It is expressed in many tissues and induced by a variety of stress agents affecting many cellular pathways. SIP generates, by alternative splicing, two nuclear proteins that can promote cell death by apoptosis.

Expression profiling in pancreas during the acute phase of pancreatitis using cDNA microarrays.

Most attacks of acute pancreatitis are self-limiting, suggesting that the pancreatic cells adapt their phenotype to prevent progression of the disease. Such phenotypic change must involve a coordinated modification in the expression of numerous genes. To identify differentially expressed genes, high-density mouse cDNA microarrays were hybridized with cDNA probes from both healthy pancreas and pancreas affected by acute pancreatitis. From the 7981 mouse genes analyzed, 239 showed significant changes in their expression during the acute phase of pancreatitis. Among them, 107 genes were up-regulated whereas 132 were down-regulated. They include genes whose function was not previously related to pancreatitis, suggesting that they are involved in some way into the acute pancreatic response. Finally, 40% of differentially expressed genes corresponded to ESTs. Demonstration that a large quantity of unexpected or yet uncharacterized genes showed altered expression during acute pancreatitis underscores the interest of a genome-based investigation. Some of these genes are certainly involved in the cellular defense against pancreatitis and, as such, deserve being studied further.

Lipopolysaccharide directly affects pancreatic acinar cells: implications on acute pancreatitis pathophysiology.

We have explored whether lipopolysaccharide (LPS, endotoxin) induces pancreatic injury on pancreatic acinar cells both in vivo and in vitro. Wistar male rats were treated with four intraperitoneal injections of 10 mg/kg LPS, and AR4-2J cells were exposed to increasing doses of LPS. Expression of pancreatitis-associated-protein (PAP) mRNA was strongly induced in AR4-2J cells exposed to LPS, while amylase mRNA was reduced. LPS also induced apoptosis and expression of TNF-alpha, IL-1beta, and IL-8 mRNA in AR4-2J cells. The in vivo effect of LPS showed structural signs of cellular damage, including numerous cytoplasmic vacuoles, severe nuclear alterations, and high expression of PAP mRNA. This study demonstrated that LPS induced pancreatic damage by directly affecting the pancreatic acinar cells. The role of LPS in the pathophysiology of acute pancreatitis may be partly due to the effect LPS has on the acinar cell.

Pancreatic acinar cells submitted to stress activate TNF-alpha gene expression.

To elucidate whether pancreatic acinar cell submitted to stress is able to express TNF-alpha, we studied TNF-alpha mRNA expression by Northern blot and in situ hybridization in healthy pancreas, in tissue from caerulein-induced pancreatitis and after lipopolysaccharide (LPS) treatment. In specimens from normal pancreas, TNF-alpha mRNA expression, as judged by both Northern blot and in situ hybridization, was negative, whereas a strong but transient expression was observed in acinar cells from caerulein pancreatitis and LPS treatment. TNF-alpha mRNA appeared as rapidly as 30 min after treatment, and was maximal 6 h after. At this time, there was mild infiltration consisting mostly of polymorphonuclear leukocytes (PMNL) and no signal of TNF-alpha transcript was found in their cytoplasm. Our results strongly indicate that pancreatic acinar cell is the source of TNF-alpha early in the course of acute pancreatitis and LPS treatment, and suggest that the expression of this cytokine is a part of a general response of the acinar cell to aggression.

Cloning and expression of the mouse PIP49 (Pancreatitis Induced Protein 49) mRNA which encodes a new putative transmembrane protein activated in the pancreas with acute pancreatitis.

We have used a microarray-based strategy to characterize, at the molecular level, the pancreatic emergency program set up by the pancreatic cells in response to pancreatitis. In this strategy, the phenotype of the pancreatitis-affected pancreas is established by characterization of a large number of its transcripts using a high-density mouse cDNA microarray. This method allows identification of transcripts differentially expressed during pancreatitis. We describe here the cloning, sequencing, and expression analysis of a new gene, named PIP49 (Pancreatitis Induced Protein 49). Its very strong expression is specific of acinar cells and occurs rapidly after initiation of the acute phase of pancreatitis. Analysis of its primary and secondary structures strongly suggests that PIP49 encodes a putative transmembrane protein.

Lipopolysaccharides induce p8 mRNA expression in vivo and in vitro.

Systemic LPS endotoxin is associated with acute pancreatic damage. Whether damage results from direct interaction of LPS with pancreatic cells is unknown. We addressed that question by monitoring p8 expression in reponse to LPS, in vivo and in vitro, because overexpression of the p8 protein is a sensitive marker of pancreatic agression. For in vivo studies, rats were sacrificed at different times after a single intraperitoneal injection of LPS, and pancreas, liver, kidney, lung, brain, and intestine were processed for RNA preparation. In vitro, pancreatic acinar AR4-2J cells were cultivated with 0.1, 1, or 10 micrograms/ml LPS for 6, 12, or 24 h. p8 mRNA expression was monitored by Northern blotting. In vivo, it was strongly increased in the pancreas after 12 h of treatment and remained elevated after 24 h. It was also induced in kidney and liver, with a maximum at 6 and 12 h, respectively, but not in lung, brain, or intestine. In AR4-2J cells, basal p8 mRNA expression was very low and increased in a time- and dose-dependent manner after treatment with LPS. LPS-induced overexpression of p8 mRNA in vivo confirmed the adverse effect of endotoxemia on pancreas and its overexpression in vitro demonstrated a direct interaction of LPS with pancreatic cells.

Clusterin overexpression in rat pancreas during the acute phase of pancreatitis and pancreatic development.

Molecular mechanisms associated with apoptosis in pancreas remain largely unknown. Clusterin mRNA is induced in several tissues in response to most apoptotic stimuli. In these tissues, clusterin has an antiapoptotic activity. The aim of this work was to test whether clusterin, which is not expressed in normal pancreas, was induced in pancreas during pancreatitis and pancreatic development. Clusterin mRNA levels were strongly increased 6 h after pancreatitis induction. Maximal expression happened between 24-48 h and decreased progressively to undetectable levels at day 5. Clusterin mRNA was expressed with similar intensity in oedematous caerulein-induced pancreatitis and in response to various degrees of necrohaemorrhagic taurocholate-induced pancreatitis, indicating a maximal gene activity in all types of pancreatitis; in situ hybridization showed that the acinar cells and some ducts expressed clusterin mRNA. A single band of about 35-38 kDa was detected by western blot in pancreatic homogenates and in pancreatic juice from rats with acute pancreatitis, but not from control rats. Clusterin mRNA expression was strong in late fetal life and remains high until day 11 post-partum, then decreased progressively with a minimum from 35 to 90 days post-partum. Clusterin mRNA levels were strongly induced in pancreatic acinar AR4-2J cells in response to various apoptotic stimuli (i.e., cycloheximide, staurosporine, ceramide and H2O2) but not with interleukin (IL)-1, IL-4 or IL-6 or heat shock, which do not induce apoptosis in AR4-2J cells. In conclusion, we demonstrated that clusterin is synthesized and released by the pancreas. Its strong expression during acute pancreatitis suggests its involvement in the pancreatic response to injury. Clusterin is also induced during pancreatic development. Because these situations are associated with apoptosis and clusterin was shown to protect against apoptosis, we speculate that clusterin could be involved in the control of acinar cell apoptosis.

The effect of chronic intraperitoneal infusion of bacterial endotoxin on exocrine pancreas function in rats.

CONCLUSION: This study demonstrated that LPS infusion can induce tissue lesions and impair the exocrine protein secretion of the pancreas in rats. BACKGROUND: The effect of chronic ip infusion of lipopolysaccharide (LPS) on the exocrine pancreas function was studies in rats. METHODS: Four milligrams per kilogram per day of Salmonella typhi LPS were infused intraperitoneally by means of surgically implanted osmotic pumps. Rats were studied after 7-d LPS infusion. RESULTS: Plasma fibrinogen and amylase activity increased significantly in LPS-treated rats when compared with control rats. Histological examination of the pancreas showed congestion, infiltration, and focal necrosis in LPS-treated rats. The pancreas wet weight, as well as DNA and total soluble protein contents were significantly increased in LPS-treated animals when compared with controls. The pancreas protein output was significantly decreased in pure pancreatic juice, whereas the pancreatic juice flow rate was significantly increased in LPS-treated animals, when compared with controls. Electrophoretic patterns showed a marked decrease in digestive enzyme contents, whereas there was an increased content of 15 kDa protein.

Effect of ethanol intake on pancreatic exocrine secretion in mice.

Swiss mice were fed conventional lab chow and 10% ethanol or water as drinking fluid for 2 weeks. Pancreatic juice was obtained by cannulation of the bile pancreatic common duct of mice anesthetized with urethane. Isolated pancreatic lobules were also obtained. The flow rate and the amylase output were determined in pure pancreatic juice. The release of amylase was measured in pancreatic lobule preparations. The basal pancreatic juice flow rate and the amylase output were significantly increased by ethanol consumption. The magnitude of the pancreatic juice flow rate and the amylase output responses to increasing doses of bethanechol, a cholinergic agent, was significantly decreased in ethanol-fed mice. The amount of spontaneously released amylase was higher in pancreatic lobule preparations from ethanol-fed animals than that from control mice, and the difference was abolished by addition of atropine to the incubation media. The amylase release rate in response to increasing doses of bethanechol was significantly reduced in lobule preparations from the ethanol-fed group. These data indicate that ethanol intake in mice has a stimulating effect on the spontaneous pancreatic secretion and lends support to the hypothesis that ethanol consumption increases the intrapancreatic cholinergic tone.

Changes in pancreatic exocrine secretion after repeated haloperidol administration.

The effect of repeated administration of haloperidol on the pancreatic secretion was studied in urethane-anesthetized Swiss mice. Haloperidol (2 mg/kg) injected daily i.p. for 7 days, increase the volume and protein content of the basal pancreatic juice significantly. This secretory activity was partially blocked by i.p. injection of atropine (5 mg/kg), both in control and treated animals. The volume of the secretory response to bethanechol, a cholinergic agonist, was decreased by haloperidol without any change in amylase release. From these findings it is concluded that repeated haloperidol treatment produces an increase of basal pancreatic secretion, which is probably the result of changes in the sensitivity of dopamine receptors of the gland.

Pure pancreatic juice in humans: orange-lemon-juice-induced secretory effects. Comparative analysis with a regular meal, sorbitol, acidified peptone broth and secretin.

The secretory effect elicited by the ingestion of 100 ml of orange-lemon juice (O.-L.J.) was studied on pure pancreatic juice obtained from a catheter placed in the human Wirsung duct at surgery. These changes were compared with those evoked by a regular meal (R.M.), the ingestion of a Sorbitol solution (S.S.), the intragastric infusion of an acidified peptone broth (A.P.B.) and an i.v. single injection of secretin (Boots, 1.0 U/kg). The O.-L.J. induced purer pancreatic secretion response (flow, bicarbonate and enzyme output) than that triggered by the R.M., S.S. and A.P.B. The O.-L.J. evoked peak values, were observed earlier (60 min) than with a R.M. (90 min) ingestion. The 120-min-cumulative values confirmed these findings and disclosed that O.-L.J. elicits a rate of secretion and bicarbonate output closely similar to that of an i.v. secretin injection and amylase response greater than that evoked by this hormone. Thus, O.-L.J. ingestion proved to be an unexpected powerful stimulus of exocrine pancreatic secretion.

Synthesis and contents of pancreatic exportable enzymes. Effect of oleic acid intraduodenal administration.

The effect of intraduodenal oleic acid administration on protein synthesis and enzymatic levels in rat pancreas was investigated. Sprague-Dawley rats were sacrificed at 20, 40, 60, and 80 min after intraduodenal oleic acid administration. Ten minutes before sacrifice, the rats were injected with 50 microCi 3H-Phenylalanine intraperitoneally. Amylase (Am), chymotrypsinogen (Chtg), trypsinogen (Tg) and lipase (Li) activities, and 3H-Phenylalanine incorporation to total secretory proteins were determined in pancreas homogenates. Forty minutes after oleic acid administration, the activities of Chtg, Tg and Li were significantly increased (45, 38 and 23%, respectively) above those from control rats. Amylase levels were not modified. Enzyme activities decreased below baseline levels by 60 and 80 min after oleic acid administration. The 3H-phenylalanine incorporation pattern exhibited a peak at 40 min. We conclude that intraduodenal oleic acid administration stimulates intrapancreatic enzyme content in a non-parallel fashion, before enzyme activities decreased below those from control rats. Protein synthesis was similarly affected by intraduodenal oleic acid.

Synthesis and contents of pancreatic exportable enzymes. Effect of oleic acid intraduodenal administration.

The effect of intraduodenal oleic acid administration on protein synthesis and enzymatic levels in rat pancreas was investigated. Sprague-Dawley rats were sacrificed at 20, 40, 60, and 80 min after intraduodenal oleic acid administration. Ten minutes before sacrifice, the rats were injected with 50 microCi 3H-Phenylalanine intraperitoneally. Amylase (Am), chymotrypsinogen (Chtg), trypsinogen (Tg) and lipase (Li) activities, and 3H-Phenylalanine incorporation to total secretory proteins were determined in pancreas homogenates. Forty minutes after oleic acid administration, the activities of Chtg, Tg and Li were significantly increased (45, 38 and 23

, respectively) above those from control rats. Amylase levels were not modified. Enzyme activities decreased below baseline levels by 60 and 80 min after oleic acid administration. The 3H-phenylalanine incorporation pattern exhibited a peak at 40 min. We conclude that intraduodenal oleic acid administration stimulates intrapancreatic enzyme content in a non-parallel fashion, before enzyme activities decreased below those from control rats. Protein synthesis was similarly affected by intraduodenal oleic acid.