Pancreatic cancer: The microenvironment needs attention too!

The abundant stromal/desmoplastic reaction, a characteristic feature of a majority of pancreatic adenocarcinomas (PDAC), has only recently been receiving some attention regarding its possible role in the pathobiology of pancreatic cancer. It is now well established that the cells predominantly responsible for producing the collagenous stroma are pancreatic stellate cells (PSCs). In addition to extracellular matrix proteins, the stroma also exhibits cellular elements including, immune cells, endothelial cells and neural cells. Evidence is accumulating to indicate the presence of significant interactions between PSCs and cancer cells as well as between PSCs and other cell types in the stroma. The majority of research reports to date, using in vitro and in vivo approaches, suggest that these interactions facilitate local growth as well as distant metastasis of pancreatic cancer, although a recent study using animals depleted of myofibroblasts has raised some questions regarding the central role of myofibroblasts in cancer progression. Nonetheless, novel therapeutic strategies have been assessed, mainly in the pre-clinical setting, in a bid to interrupt stromal-tumour interactions and inhibit disease progression. The next important challenge is for the translation of such pre-clinical strategies to the clinical situation so as to improve the outcome of patients with pancreatic cancer.

A fire inside: current concepts in chronic pancreatitis.

Chronic pancreatitis is a necroinflammatory process characterized pathologically by acinar atrophy and fibrosis and clinically by abdominal pain, diabetes and maldigestion. In this review we summarize some of the recent advances in the understanding of the pathogenesis of pancreatitis and how they have shaped our current understanding of chronic pancreatitis. We pay particular attention to advances in the genetic basis of idiopathic, hereditary and tropical pancreatitis as well as research into the relationship between alcohol and the pancreas. We have also reviewed current practices with respect to diagnosis and management of chronic pancreatitis.

Chronic ethanol administration decreases rat pancreatic GP2 content.

Postulated mechanisms of alcoholic pancreatitis include (i) zymogen granule fragility facilitating intracellular activation of digestive enzymes and (ii) ductular obstruction by protein plugs. GP2, a pancreatic glycoprotein, stabilizes zymogen granule membranes and is an important constituent of pancreatic protein plugs. Therefore, this study examined the pancreatic content and messenger RNA levels of GP2 after chronic ethanol administration. Rats were fed liquid diets with or without ethanol, for four weeks. GP2 levels in pancreatic homogenates, crude zymogen granules and zymogen granule membrane fractions were assessed by immunoblotting. Messenger RNA levels for GP2 were measured by Northern and dot blotting of pancreatic RNA. Pancreatic GP2 levels were lower in ethanol-fed rats than in controls (GP2 levels expressed as % of control: 38.75 +/- 5.8, p < 0.001 in homogenate; 31.28 +/- 3.5, p < 0.0005 in crude zymogen granules and 22.89 +/- 5.4, p < 0.0005 in zymogen granule membranes). Messenger RNA levels for GP2 were unchanged after ethanol feeding. Chronic ethanol consumption decreases GP2 content of pancreatic homogenate and zymogen granules. This decrease could (i) result from an increased release into pancreatic juice thereby favouring protein plug formation and (ii) impair zymogen granule stability. Both these mechanisms could potentiate pancreatic damage.

Alcohol-induced pancreatic injury.

Alcoholic pancreatitis is a major complication of alcohol abuse. Until recently, it was generally accepted that alcoholic pancreatitis was a chronic disease from the outset. However, evidence is now emerging in support of the 'necrosis-fibrosis' hypothesis that alcoholic pancreatitis begins as an acute process and that repeated episodes of acute injury lead to the changes of chronic pancreatitis (acinar atrophy and fibrosis) resulting in exocrine and endocrine dysfunction. The treatment of acute pancreatitis follows the regimen of bed rest, nasogastric suction, analgesia and intravenous support. The role of additional therapeutic measures such as prophylactic antibiotics, antioxidants and enteral nutrition in severe cases has not yet been precisely defined. The treatment of chronic pancreatitis involves attention to its three cardinal features: pain, maldigestion and diabetes. With respect to the pathogenesis of alcoholic pancreatitis, the focus of research over the past 30 years has shifted from the sphincter of Oddi and ductular abnormalities to the acinar cell itself. It has now been established that the acinar cell is capable of metabolizing alcohol and that direct toxic effects of alcohol and/or its metabolites on acinar cells may predispose the gland to injury in the presence of an appropriate trigger factor. A significant recent development relates to the characterization of pancreatic stellate cells, increasingly implicated in alcoholic pancreatic fibrosis. This chapter summarizes the natural history, clinical features, current trends in treatment as well as recent advances in our understanding of the pathogenesis of alcoholic pancreatitis.

Alcohol-related pancreatic damage: mechanisms and treatment.

Pancreatitis is a potentially fatal inflammation of the pancreas often associated with long-term alcohol consumption. Symptoms may result from blockage of small pancreatic ducts as well as from destruction of pancreatic tissue by digestive enzymes. In addition, by-products of alcohol metabolism within the pancreas may damage cell membranes. Research on the causes of pancreatitis may support more effective disease management and provide hope for a potential cure.

Both ethanol and protein deficiency increase messenger RNA levels for pancreatic lithostathine.

Both ethanol abuse and protein deficiency are well known associations of chronic pancreatitis. An early event in chronic pancreatitis is the deposition of protein plugs in small pancreatic ducts, leading to ductular obstruction and acinar cell damage. Lithostathine, a pancreatic secretory protein, is a major organic component of protein plugs. The aim of this study was to determine the effect of chronic ethanol administration and dietary protein deficiency, separately and in combination, on messenger RNA (mRNA) levels for pancreatic lithostathine. Male Sprague-Dawley rats were fed in groups of four, for four weeks, protein sufficient and protein deficient diets with or without ethanol. Messenger RNA levels for pancreatic lithostathine were assessed in all four groups. Both ethanol and protein deficiency, separately and in combination, increased mRNA levels for lithostathine. Thus, both chronic ethanol consumption and dietary protein deficiency increase the capacity of the pancreatic acinar cell to synthesize lithostathine.

The role of inflammatory and parenchymal cells in acute pancreatitis.

The infiltration of inflammatory cells into the pancreas is an early and central event in acute pancreatitis that promotes local injury and systemic complications of the disease. Recent research has yielded the important finding that resident cells of the pancreas (particularly acinar and pancreatic stellate cells) play a dynamic role in leukocyte attraction via secretion of chemokines and cytokines and expression of adhesion molecules. Significant progress has been made in recent years in our understanding of the role of leukocyte movement (adhesion to the blood vessel wall, transmigration through the blood vessel wall and infiltration into the parenchyma) in the pathophysiology of acute pancreatitis. This review discusses recent studies and describes the current state of knowledge in the field. It is clear that detailed elucidation of the numerous processes in the inflammatory cascade is an essential step towards the development of improved therapeutic strategies in acute pancreatitis. Studies to date suggest that combination therapy targeting different steps of the inflammatory cascade may be the treatment of choice for this disease.

Vitamin A inhibits pancreatic stellate cell activation: implications for treatment of pancreatic fibrosis.

BACKGROUND AND AIMS: Activated pancreatic stellate cells (PSCs) are implicated in the production of alcohol induced pancreatic fibrosis. PSC activation is invariably associated with loss of cytoplasmic vitamin A (retinol) stores. Furthermore, retinol and ethanol are known to be metabolised by similar pathways. Our group and others have demonstrated that ethanol induced PSC activation is mediated by the mitogen activated protein kinase (MAPK) pathway but the specific role of retinol and its metabolites all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-RA) in PSC quiescence/activation, or its influence on ethanol induced PSC activation is not known. Therefore, the aims of this study were to (i) examine the effects of retinol, ATRA, and 9-RA on PSC activation; (ii) determine whether retinol, ATRA, and 9-RA influence MAPK signalling in PSCs; and (iii) assess the effect of retinol supplementation on PSCs activated by ethanol. METHODS: Cultured rat PSCs were incubated with retinol, ATRA, or 9-RA for varying time periods and assessed for: (i) proliferation; (ii) expression of alpha smooth muscle actin (alpha-SMA), collagen I, fibronectin, and laminin; and (iii) activation of MAPKs (extracellular regulated kinases 1 and 2, p38 kinase, and c-Jun N terminal kinase). The effect of retinol on PSCs treated with ethanol was also examined by incubating cells with ethanol in the presence or absence of retinol for five days, followed by assessment of alpha-SMA, collagen I, fibronectin, and laminin expression. RESULTS: Retinol, ATRA, and 9-RA significantly inhibited: (i) cell proliferation, (ii) expression of alpha-SMA, collagen I, fibronectin, and laminin, and (iii) activation of all three classes of MAPKs. Furthermore, retinol prevented ethanol induced PSC activation, as indicated by inhibition of the ethanol induced increase in alpha-SMA, collagen I, fibronectin, and laminin expression. CONCLUSIONS: Retinol and its metabolites ATRA and 9-RA induce quiescence in culture activated PSCs associated with a significant decrease in the activation of all three classes of MAPKs in PSCs. Ethanol induced PSC activation is prevented by retinol supplementation.

Molecular mechanisms of alcoholic pancreatitis.

Alcoholic pancreatitis is a major complication of alcohol abuse. Since only a minority of alcoholics develop pancreatitis, there has been a keen interest in identifying the factors that may confer individual susceptibility to the disease. Numerous possibilities have been evaluated including diet, drinking patterns and a range of inherited factors. However, at the present time, no susceptibility factor has been unequivocally identified. In contrast, considerable progress has been made with respect to the constant effects of alcohol on the pancreas. The molecular mechanisms of alcohol-induced pancreatic injury are being increasingly defined with an emphasis, in recent years, on the acinar cell itself as the principal site on ethanol-related damage. It has now been established that the acinar cell is capable of metabolizing alcohol and that the direct toxic effects of alcohol and/or its metabolites on acinar cells may predispose the gland to autodigestive injury in the presence of an appropriate triggering factor. A significant recent development relates to the characterization of pancreatic stellate cells, increasingly implicated in alcoholic pancreatic fibrosis. Here the current concepts regarding the mechanisms/pathways mediating alcohol-induced pancreatic injury are outlined.

Role of alcohol metabolism in alcoholic pancreatitis.

Metabolism of ethanol by acinar and other pancreatic cells and the consequent generation of toxic metabolites are postulated to play an important role in the development of alcohol-related acute and chronic pancreatic injury. Studies using cultured pancreatic acinar cells and isolated pancreatic acini have established that (i) the pancreas can metabolize ethanol via the oxidative pathway involving the enzymes alcohol dehydrogenase (ADH) and possibly cytochrome P4502E1 (although the role of the latter remains to be fully delineated) as well as the nonoxidative pathway [involving fatty acid ethyl ester (FAEE) synthases] and (ii) the oxidative pathway (which generates acetaldehyde) is quantitatively greater than the nonoxidative pathway, which yields FAEEs. Most recently, pancreatic stellate cells (PSCs) (implicated in pancreatic fibrogenesis) have been reported to exhibit ADH activity, suggesting that the capacity of the pancreas to metabolize ethanol may reside not only in parenchymal (acinar) cells but also in nonparenchymal cells. Polymorphisms/mutations of ethanol metabolizing enzymes have been examined to determine whether they may confer individual susceptibility to alcoholic pancreatitis. However, no association has been demonstrated between ADH and CYP2E1 polymorphisms and the predisposition to alcoholic pancreatitis. Other candidate factors that remain to be studied include polymorphisms of FAEE synthetic enzymes and proteins relevant to antioxidant pathways in the cell. Injury to the pancreas due to its capacity to metabolize ethanol may be mediated by direct effects of both acetaldehyde and FAEEs and by alterations induced within the cells during ethanol metabolism, such as changes in the intracellular redox state and the generation of oxidant stress.

Stellate cell activation in alcoholic pancreatitis.

The pathogenesis of pancreatic fibrosis, a characteristic feature of alcohol-induced chronic pancreatitis, has received increasing attention over the past few years, largely due to the identification and characterization of stellate cells in the pancreas. These cells are morphologically similar to hepatic stellate cells, the principal effector cells in liver fibrosis. The role of pancreatic stellate cells (PSCs) in alcoholic pancreatic fibrosis has been studied using 2 approaches: (i) in vivo studies using pancreatic tissue from patients with alcohol-induced chronic pancreatitis and from animal models of experimental pancreatitis and (ii) in vitro studies using cultured PSCs. These studies indicate that PSCs are activated early in the course of pancreatic injury and are the predominant source of collagen in the fibrotic pancreas. Several factors that may be responsible for mediating PSC activation during chronic alcohol exposure have also been identified. From the findings to date, it may be speculated that the pathogenesis of alcoholic pancreatic fibrosis may involve 2 pathways: (i) a necroinflammatory pathway involving cytokine release and PSC activation and (ii) a nonnecroinflammatory pathway involving direct activation of PSCs by ethanol via its metabolism to acetaldehyde and the generation of oxidant stress.

Mechanisms of pancreatic fibrosis.

Pancreatic fibrosis, a characteristic histopathological feature of chronic pancreatitis, is no longer considered an epiphenomenon of chronic injury, but an active process that may be reversible in the early stages. The identification and characterization of pancreatic stellate cells (PSCs) in recent years has had a significant impact on research into pancreatic fibrogenesis. Accumulating evidence from both in vivo studies (using human pancreatic sections and experimental models of pancreatic fibrosis) and in vitro studies (using cultured pancreatic stellate cells) indicates a key role for activated PSCs in the fibrotic process. These cells are now known to be activated by ethanol and its metabolites and by several factors that are upregulated during pancreatic injury including growth factors, cytokines and oxidant stress. Based on this knowledge, potential antifibrotic strategies such as antioxidants and cytokine inhibition have been assessed in experimental models. Studies are also underway to characterise the signaling pathways/molecules responsible for mediating PSC activation, in order to identify potential therapeutic targets for the inhibition of PSC activation, thereby preventing or reversing the development of pancreatic fibrosis.

Ethanol induced acinar cell injury.

Ethanol abuse is a well known association of pancreatitis. Research into the pathogenesis of alcoholic pancreatitis has generally followed two directions. Firstly, factors which may predispose alcoholics to pancreatitis have been examined. To date, these studies have been negative and the predisposing factor(s) remain unknown. The second approach has involved studies on the constant metabolic effects of ethanol on the pancreas which may render the acinar cell susceptible to digestive enzyme induced injury. Recently developed models of experimental pancreatitis have implicated intracellular activation of digestive enzymes by lysosomal enzymes as an early event. Using the Lieber-DeCarli model of ethanol administration to rats, a number of changes have been described in pancreatic acinar cells which may predispose the gland to autodigestion. These changes include: (1) increased glandular content of digestive enzymes as a result of increases in mRNA levels for these enzymes; (2) increased glandular content of the lysosomal enzyme cathepsin B (known to be capable of activating trypsinogen); (3) increased fragility of lysosomes possibly mediated by cholesteryl esters and fatty acid ethyl esters; and (4) increased fragility of zymogen granules. These effects of ethanol constitute a "primed" setting (the "Drinker's Pancreas") for autodigestion. Triggering factors for autodigestion in this setting have not yet been identified.

Chronic pancreatitis: complications and management.

Chronic pancreatitis is characterized by progressive and irreversible loss of pancreatic exocrine and endocrine function. In the majority of cases, particularly in Western populations, the disease is associated with alcohol abuse. The major complications of chronic pancreatitis include abdominal pain, malabsorption, and diabetes. Of these, pain is the most difficult to treat and is therefore the most frustrating symptom for both the patient and the physician. While analgesics form the cornerstone of pain therapy, a number of other treatment modalities (inhibition of pancreatic secretion, antioxidants, and surgery) have also been described. Unfortunately, the efficacy of these modalities is difficult to assess, principally because of the lack of properly controlled clinical trials. Replacement of pancreatic enzymes (particularly lipase) in the gut is the mainstay of treatment for malabsorption; the recent discovery of a bacterial lipase (with high lipolytic activity and resistance to degradation in gastric and duodenal juice) represents an important advance that may significantly increase the efficacy of enzyme replacement therapy by replacing the easily degradable porcine lipase found in existing enzyme preparations. Diabetes secondary to chronic pancreatitis is difficult to control and its course is often complicated by hypoglycaemic attacks. Therefore, it is essential that caution is exercised when treating this condition with insulin. This paper reviews recent research and prevailing concepts regarding the three major complications of chronic pancreatitis noted above. A comprehensive discussion of current opinion on clinical issues relating to the other known complications of chronic pancreatitis such as pseudocysts, venous thromboses, biliary and duodenal obstruction, biliary cirrhosis, and pancreatic cancer is also presented.

Fatty acid ethyl esters increase rat pancreatic lysosomal fragility.

Recent studies indicate that altered lysosomal function may be involved in the early stages of pancreatic injury. Chronic consumption of ethanol has been shown to increase rat pancreatic lysosomal fragility. Fatty acid ethyl esters (nonoxidative products of ethanol metabolism) accumulate in the pancreas after ethanol consumption. The aim of this study was to determine whether the lysosomal fragility observed after ethanol could be mediated by fatty acid ethyl esters. Rat pancreatic lysosomes were incubated for 20 minutes at 20 degrees C with ethyl oleate (a representative fatty acid ethyl ester). Lysosomal stability was then assessed by determination of (1) latency (i.e., the percent increase in lysosomal enzyme activity after addition of Triton X-100) and (2) supernatant activity (i.e., the proportion of lysosomal enzyme remaining in the supernatant after resedimentation of lysosomes). N-acetyl glucosaminidase and cathepsin B were assayed as lysosomal marker enzymes. Lysosomes incubated with buffer alone were used as controls. Ethyl oleate at concentrations above 140 mumol/L increased pancreatic lysosomal fragility as demonstrated by decreased latency. Increased percentage of enzyme in the supernatant was observed at higher concentrations. These results suggest that increased pancreatic lysosomal fragility observed with ethanol may be mediated by fatty acid ethyl esters.

Alcohol and the pancreas.

Alcoholic pancreatitis is a major, often lethal complication of alcohol abuse. Until recently it was generally accepted that alcoholic pancreatitis was a chronic disease from the outset. However, there is now emerging evidence in favour of the necrosis-fibrosis hypothesis that alcoholic pancreatitis begins as an acute process and that repeated acute attacks lead to chronic pancreatitis, resulting in exocrine and endocrine failure. Over the past 10-15 years, the focus of research into the pathogenesis of alcoholic pancreatitis has shifted from possible sphincteric and ductular abnormalities to the acinar cell itself which has increasingly been implicated as the initial site of injury. Recent studies have shown that the acinar cell can metabolize alcohol at rates comparable to those observed in hepatocytes. In addition, it has been demonstrated that alcohol and its metabolites exert direct effects on the pancreatic acinar cell which may promote premature digestive enzyme activation and oxidant stress. The challenge remains to identify predisposing and triggering factors in this disease.

Chronic ethanol consumption increases the fragility of rat pancreatic zymogen granules.

Intracellular activation of pancreatic digestive enzymes by lysosomal hydrolases is thought to be an early event in the pathogenesis of pancreatic injury. As ethanol excess is an important association of pancreatitis, experimental work has been directed towards exploring possible mechanisms whereby ethanol may facilitate contact between inactive digestive enzyme precursors and lysosomal enzymes. The aim of this study was to find out if chronic ethanol administration increases the fragility of rat pancreatic zymogen granules. Sixteen male Sprague-Dawley rats were pair fed ethanol and control liquid diets for four weeks. Zymogen granule fragility was then assessed in pancreatic homogenate by determination of (a) latency and (b) per cent supernatant enzyme after sedimentation of zymogen granules. Amylase was used as a zymogen granule marker enzyme. Latency was significantly reduced in pancreatic homogenates of ethanol fed animals suggesting increased zymogen granule fragility. In support of this finding, there was a trend towards increased supernatant enzyme after ethanol feeding. In conclusion, administration of ethanol increases the fragility of pancreatic zymogen granules in the absence of morphological evidence of pancreatic injury. It is proposed that zymogen granule fragility may play an early part in the pathogenesis of alcoholic pancreatitis by permitting contact between digestive and lysosomal enzymes.

Lipid intolerance does not account for susceptibility to alcoholic and gallstone pancreatitis.

BACKGROUND/AIMS: Hypertriglyceridemia is an established cause of pancreatitis and has been suggested as a predisposing factor in alcohol and gallstone-induced pancreatitis. The aims of this study were to determine fasting and postprandial triglyceride levels of alcoholics with pancreatitis, alcoholics without pancreatitis, patients with previous gallstone pancreatitis, patients with choledocholithiasis, and healthy controls. METHODS: Oral lipid tolerance studies were performed in the above groups. RESULTS: No relationship was found between alcoholic pancreatitis and hypertriglyceridemia, regardless of whether subjects were studied in the fasting state, after ingestion of fat, or after ingestion of fat with ethanol. Plasma triglyceride levels of alcoholics with pancreatitis remained similar to those of alcoholics without pancreatitis, but levels in both groups varied in relation to recent alcohol intake. Plasma triglyceride levels from both groups of alcoholics were greater than those of nonalcoholic healthy subjects. In addition, the previously reported association between postprandial hypertriglyceridemia and gallstone pancreatitis was not observed. CONCLUSIONS: Plasma triglyceride levels do not account for individual susceptibility to either alcoholic or gallstone pancreatitis.

Effects of ethanol and protein deficiency on pancreatic digestive and lysosomal enzymes.

The pathogenesis of alcoholic pancreatitis is not fully understood. An increase in pancreatic digestive and lysosomal enzyme synthesis because of ethanol consumption could contribute to the development of pancreatic injury in alcoholics. This study aimed, firstly, to determine the effect of ethanol on the content and messenger RNA levels of pancreatic digestive enzymes and on the messenger RNA level of the lysosomal enzyme cathepsin B, and secondly, to examine the influence of concomitant protein deficiency (a known association of alcoholism and pancreatic injury) on these effects. A rat model of chronic ethanol administration was used in which rats were fed in groups of four, and for four weeks, protein sufficient and protein deficient diets with or without ethanol. Ethanol increased the pancreatic content of lipase but did not influence chymotrypsinogen or trypsinogen values. mRNA levels for lipase, trypsinogen, and chymotrypsinogen were raised in rats fed ethanol. Protein deficiency resulted in reduced tissue levels of lipase, chymotrypsinogen, and amylase but did not influence trypsinogen values. mRNA levels for proteases were increased in protein deficient rats, while those for lipase remained unaltered. Both ethanol and protein deficiency increased mRNA levels for cathepsin B. It is concluded that chronic ethanol consumption, in both protein sufficient and protein deficient states, increases the capacity of the pancreatic acinar cell to synthesise digestive and lysosomal enzymes.

Both ethanol consumption and protein deficiency increase the fragility of pancreatic lysosomes.

Both ethanol abuse and protein deficiency result in pancreatic injury. Moreover, these two variables frequently coexist. As lysosomal enzymes may play a role in the initiation of pancreatic injury, the aim of this study was to determine the effects of ethanol consumption and protein deficiency on pancreatic lysosomal stability. For 3 weeks, male Sprague-Dawley rats were match-fed (in groups of four) isocaloric amounts of one of the following liquid diets: (1) protein-sufficient diet, (2) protein-sufficient diet containing ethanol as 36% of the total energy, (3) protein-deficient diet, and (4) protein-deficient diet containing ethanol as 36% of energy. Pancreatic lysosomal stability was assessed by determining (a) latency, as indicated by the percentage increase in lysosomal enzyme activity in pancreatic homogenate induced by Triton X-100, and (b) by the percentage of lysosomal enzyme remaining in the supernatant after sedimentation of the lysosomal pellet from the pancreatic homogenate. Protein deficiency was associated with a decrease in latency and an increase in supernatant enzyme. Ethanol administration was associated with a decreased latency. Both protein-deficient and ethanol-fed animals exhibited higher pancreatic activities of cathepsin B, a lysosomal protease capable of activating trypsinogen. In addition, protein-deficient animals exhibited higher pancreatic activities of acid phosphatase, N-acetyl-glucosaminidase, and beta-glucuronidase. As lysosomal enzymes are postulated to play a role in the initiation of pancreatitis, these results suggest that ethanol consumption and protein deficiency may at least partly exert their toxic effects on the pancreas by altering pancreatic lysosomal stability and increasing the glandular content of cathepsin B.