Pancreatic cancer carries a poor prognosis as most patients present with advanced disease and preferred chemotherapy regimens offer only modest effects on survival. Risk factors include smoking, obesity, heavy alcohol, and chronic pancreatitis. Pancreatic cancer has a complex relationship with diabetes, as diabetes can be both a risk factor for pancreatic cancer and a result of pancreatic cancer. Insulin, insulin-like growth factor-1 (IGF-1), and certain hormones play an important role in promoting neoplasia in diabetics. Metformin appears to reduce risk for pancreatic cancer and improve survival in diabetics with pancreatic cancer primarily by decreasing insulin/IGF signaling, disrupting mitochondrial respiration, and inhibiting the mammalian target of rapamycin (mTOR) pathway. Other potential anti-tumorigenic effects of metformin include the ability to downregulate specificity protein transcription factors and associated genes, alter microRNAs, decrease cancer stem cell proliferation, and reduce DNA damage and inflammation. Here, we review the most recent knowledge on risk factors and treatment of pancreatic cancer and the relationship between diabetes, pancreatic cancer, and metformin as a potential therapy.
metformin; pancreatic cancer; diabetes; mTOR; AMPK; insulin; IGF-1
Ellagic acid is a polyphenolic phytochemical present in many fruits and nuts with anti-cancer properties demonstrated in experimental tumor studies. Embelin is a benzoquinone phytochemical isolated from the Japanese herb Ardisiae Japonicae and has been shown to induce apoptosis in cancer cells. We found that ellagic acid and embelin each dose-dependently increased apoptosis and inhibited proliferation in human pancreatic cancer cells, MIA PaCa-2 and HPAF-II cells, and in pancreatic stellate cells (PaSCs) which are progenitors of pancreatic cancer desmoplasia. In each of these cell types, combinations of ellagic acid and embelin at low micromolar concentrations (0.5–3 μM) induced synergistic increases in apoptosis and decreases in proliferation. Ellagic acid decreased NF-κB transcriptional activity, whereas embelin decreased STAT-3 phosphorylation and protein expression of its downstream target survivin, in cancer cells. In vivo dietary ellagic acid alone or in combination with embelin decreased tumor size and tumor cellularity in a subcutaneous (s.c.) xenograft mouse model of pancreatic cancer. These results show that ellagic acid and embelin interact with divergent intracellular signaling pathways resulting in augmentation of apoptosis and inhibition of proliferation at low micromolar concentrations for the key cellular components of pancreatic adenocarcinoma.
Ellagic acid; Embelin; pancreatic cancer; apoptosis; proliferation
There is epidemiologic evidence that obesity increases the risk of cancers. Several underlying mechanisms, including inflammation and insulin resistance, are proposed. However, the driving mechanisms in pancreatic cancer are poorly understood. The goal of the present study was to develop a model of diet-induced obesity and pancreatic cancer development in a state-of-the-art mouse model, which resembles important clinical features of human obesity, e.g. weight gain and metabolic disturbances.
Offspring of Pdx-1-Cre and LSL-KrasG12D mice were allocated to either a diet high in fats and calories (HFCD; ~4,535 kcal/kg; 40% of calories from fats) or control diet (CD; ~3,725 kcal/kg; 12% of calories from fats) for 3 months. Compared to control animals, mice fed the HFCD significantly gained more weight and developed hyperinsulinemia, hyperglycemia, hyperleptinemia, and elevated levels of IGF-1. The pancreas of HFCD-fed animals showed robust signs of inflammation with increased numbers of infiltrating inflammatory cells (macrophages and T-cells), elevated levels of several cytokines and chemokines, increased stromal fibrosis, and more advanced PanIN lesions.
Our results demonstrate that a diet high in fats and calories leads to obesity and metabolic disturbances similar to humans and accelerates early pancreatic neoplasia in the conditional KrasG12D mouse model. This model and findings will provide the basis for more robust studies attempting to unravel the mechanisms underlying the cancer-promoting properties of obesity as well as to evaluate dietary- and chemo-preventive strategies targeting obesity-associated pancreatic cancer development.
Obesity; inflammation; pancreatic intraepithelial neoplasia; tumor promotion; mouse model
Pancreatic ductal adenocarcinoma is a devastating disease, and patient outcomes have not improved in decades. Treatments that target tumor cells have largely failed. This could be because research has focused on cancer cells and the influence of the stroma on tumor progression has been largely ignored. The focus of pancreatic cancer research began to change with the identification of pancreatic stellate cells, which produce the pancreatic tumor stroma. There is compelling in vitro and in vivo evidence for the influence of pancreatic stellate cells on pancreatic cancer development; several recent preclinical studies have reported encouraging results with approaches designed to target pancreatic stellate cells and the stroma. We review the background and recent advances in these areas, along with important areas of future research that could improve therapy.
Pancreatic Stellate Cells; Pancreatic Cancer; Inflammation; Immune Surveillance
Rottlerin is a natural polyphenolic ketone isolated from the pericarps of Mallotus phillippinensis. In previous studies we showed that parenteral administration of rottlerin reduced tumor growth in murine xenograft models of pancreatic cancer. The aim of this study was to develop a simple and validated method for the quantitative determination of rottlerin in plasma and tumor tissues of mice fed a rottlerin diet. A xenograft model of pancreatic cancer was prepared by injection of 2×106 HPAF-II cells subcutaneously into nude mice. One week before tumor implantation, mice were randomly allocated to standard diet (AIN76A) and standard diet supplement with 0.012% rottlerin (n=6 per group). Mice were sacrificed after 6 weeks on diets. Rottlerin was extracted from the plasma and tissues using protein precipitation-extraction and analyzed by reverse-phase HPLC-DAD method. The same HPLC method was also applied to determine rottlerin levels in conditioned culture media and in cell lysates from HPAF-II cells exposed to 25 µM concentration of rottlerin. A substantial amount of rottlerin was detected in tumor (2.11 ± 0.25 nmol/g tissue) and plasma (2.88 ± 0.41 µM) in mice fed rottlerin diet. In addition, significant levels of rottlerin (57.4 ± 5.4 nmol/mg protein) were detected in cell lysates from rottlerin-treated HPAF-II cells. These data indicate that rottlerin is efficiently absorbed in cells and tissues both in vivo and in vitro and suggest a strong potential for rottlerin as a preventive or adjuvant supplement for pancreatic cancer.
Rottlerin; Tissue distribution; HPLC; Pancreatic cancer; In vivo; Cell uptake
There are approximately 277 000 new cases of pancreatic cancer and 266 000 deaths from pancreatic cancer annually, indicating a mortality rate of 96% of the cases diagnosed. Because of the ineffectiveness of therapies, a major emphasis needs to be placed on prevention. This paper reviews the epidemiology and risk factors for pancreatic cancer, and uses this information to propose plausible research directions for determining the biological mechanisms mediating the effects of risk factors on the promotion of pancreatic cancer, with a focus on the pancreatic stellate cell.
diabetes; obesity; pancreatic cancer; smoking; stellate cell
Background & Aims
Mechanisms underlying synergistic liver injury caused by alcohol and obesity are not clear. We have produced a mouse model of synergistic steatohepatitis by recapitulating the natural history of the synergism seen in patients for mechanistic studies.
Moderate obesity was induced in mice by 170% overnutrition in calories using intragastric overfeeding of high fat diet. Alcohol (low or high dose) was then co-administrated to determine its effects.
Moderate obesity plus alcohol intake causes synergistic steatohepatitis in an alcohol dose-dependent manner. A heightened synergism is observed when a high alcohol dose (32g/kg/day) is used, resulting in plasma ALT reaching 392 ± 28 U/L, severe steatohepatitis with pericellular fibrosis, marked M1 macrophage activation, a 40-fold induction of iNos, and intensified nitrosative stress in the liver. Hepatic expression of genes for mitochondrial biogenesis and metabolism are significantly downregulated, and hepatic ATP level is decreased. Synergistic ER stress evident by elevated XBP-1, GRP78 and CHOP, is accompanied by hyperhomocysteinemia. Despite increased caspase 3/7 cleavage, their activities are decreased in a redox-dependent manner. Neither increased PARP cleavage nor TUNEL positive hepatocytes are found, suggesting a shift of apoptosis to necrosis. Surprisingly, the synergism mice have increased plasma adiponectin and hepatic p-AMPK, but adiponectin resistance is shown downstream of p-AMPK.
Nitrosative stress mediated by M1 macrophage activation, adiponectin resistance, accentuated ER and mitochondrial stress underlie potential mechanisms for synergistic steatohepatitis caused by moderate obesity and alcohol.
synergistic steatohepatitis; obesity and alcohol synergism; apoptosis; necrosis; macrophage activation; nitrosative stress
Alcohol abuse is the leading etiologic factor of pancreatitis, although many heavy drinkers do not develop pancreatic damage. Alcohol promotes pancreatitis through a combination of remote (e.g., increased gut permeability to bacterial products such as lipopolysaccharide) and more proximal effects (e.g., altered pancreatic cholinergic inputs), including oxidative damage at the level of the pancreatic acinar cell. Recent evidence indicates that alcohol exposure to rodents disturbs proteostasis in the exocrine pancreas, an effect counterbalanced by homeostatic processes that include both the unfolded protein response (UPR) and autophagy. A corollary to this notion is that pancreatitis results when adaptive responses are insufficiently robust to alleviate the cellular stress caused by alcohol.
acinar cell; alcohol abuse; pancreas; ERAD; UPR; XBP1
Alcohol abuse is one of the most common causes of pancreatitis. The risk of developing alcohol-induced pancreatitis is related to the amount and duration of drinking. However, only a small portion of heavy drinkers develop disease, indicating that other factors (genetic, environmental or dietary) contribute to disease initiation. Epidemiologic studies suggest roles for cigarette smoking and dietary factors in the development of alcoholic pancreatitis. The mechanisms underlying alcoholic pancreatitis are starting to be understood. Studies from animal models are revealing that alcohol sensitizes the pancreas to key pathobiologic processes that are involved in pancreatitis. Current studies are focused on the mechanisms responsible for the sensitizing effect of alcohol; recent findings reveal disordering of key cellular organelles including endoplasmic reticulum, mitochondria, and lysosomes. As our understanding of alcohol’s effects continue to advance to the level of molecular mechanisms, insights into potential therapeutic strategies will emerge providing opportunities for clinical benefit.
Alcohol abuse is a common cause of both acute and chronic pancreatitis. There is a wide spectrum of pancreatic manifestations in heavy drinkers from no apparent disease in most individuals to acute inflammatory and necrotizing pancreatitis in a minority of individuals with some progressing to chronic pancreatitis characterized by replacement of the gland by fibrosis and chronic inflammation. Both smoking and African-American ethnicity are associated with increased risk of alcoholic pancreatitis. In this review we describe how our recent studies demonstrate that ethanol feeding in rodents causes oxidative stress in the endoplasmic reticulum (ER) of the digestive enzyme synthesizing acinar cell of the exocrine pancreas. This ER stress is attenuated by a robust unfolded protein response (UPR) involving X-box binding protein-1 (XBP1) in the acinar cell. When the UPR activation is prevented by genetic reduction in XBP1, ethanol feeding causes significant pathological responses in the pancreas. These results suggest that the reason most individuals who drink alcohol heavily do not get significant pancreatic disease is because the pancreas mounts an adaptive UPR to attenuate the ER stress that ethanol causes. We hypothesize that disease in the pancreas results when the UPR is insufficiently robust to alleviate the ER stress caused by alcohol abuse.
Pancreas; Pancreatitis; Unfolded protein response; Alcohol; Smoking
BACKGROUND & AIMS
Endoplasmic reticulum (ER) stress responses (collectively known the unfolded protein response, UPR) have important roles in several human disorders, but their contribution to alcoholic pancreatitis is not known. We investigated the role of X box-binding protein 1 (XBP1), an UPR regulator, in prevention of alcohol-induced ER stress in the exocrine pancreas.
Wild-type and Xbp1+/− mice were fed control or ethanol diets for 4 weeks. Pancreatic tissue samples were then examined by light and electron microscopy to determine pancreatic alterations; UPR regulators were analyzed biochemically.
In wild-type mice, ethanol activated a UPR, increasing pancreatic levels of XBP1 and XBP1 targets such as protein disulfide isomerase (PDI). In these mice, pancreatic damage was minor. In ethanol-fed Xbp1+/− mice, XBP1 and PDI levels were significantly lower than in ethanol-fed, wild-type mice. The combination of XBP1 deficiency and ethanol feeding reduced expression of regulators of ER function and the upregulation of pro-apoptotic signals. Moreover, ethanol feeding induced oxidation of PDI, which might compromise PDI-mediated disulfide bond formation during ER protein folding. In ethanol-fed Xbp1+/− mice, ER stress was associated with disorganized and dilated ER, loss of zymogen granules, accumulation of autophagic vacuoles, and increased acinar cell death.
Chronic ethanol feeding causes oxidative ER stress, which activates a UPR and increases XBP1 levels and activity. A defective UPR, due to XBP1 deficiency, results in ER dysfunction and acinar cell pathology.
alcohol disease; transcription factors; ER protein folding; organelle
The mechanisms initiating pancreatitis in patients with chronic alcohol abuse are poorly understood. Although alcohol feeding has been previously suggested to alter cholinergic pathways, the effects of these cholinergic alterations in promoting pancreatitis have not been characterized. For the present study, we determined the role of the cholinergic system in ethanol-induced sensitizing effects on cerulein pancreatitis.
Rats were pair fed control and ethanol-containing Lieber-DeCarli diets for 6 weeks followed by parenteral administration of four hourly intraperitoneal injections of the cholecystokinin analogue, cerulein at 0.5 μg/Kg. This dose of cerulein was selected because it caused pancreatic injury in ethanol-fed but not in control-fed rats. Pancreatitis was preceded by treatment with the muscarinic receptor antagonist atropine or by bilateral subdiaphragmatic vagotomy. Measurement of pancreatic pathology included serum lipase activity, pancreatic trypsin and caspase-3 activities, and markers of pancreatic necrosis, apoptosis and autophagy. In addition, we measured the effects of ethanol feeding on pancreatic acetylcholinesterase activity and pancreatic levels of the muscarinic acetylcholine receptors m1 and m3. Finally, we examined the synergistic effects of ethanol and carbachol on inducing acinar cell damage.
We found that atropine blocked almost completely pancreatic pathology caused by cerulein administration in ethanol fed rats, while vagotomy was less effective. Ethanol feeding did not alter expression levels of cholinergic muscarinic receptors in the pancreas but significantly decreased pancreatic acetylcholinesterase activity, suggesting that acetylcholine levels and cholinergic input within the pancreas can be higher in ethanol fed rats. We further found that ethanol treatment of pancreatic acinar cells augmented pancreatic injury responses caused by the cholinergic agonist, carbachol.
These results demonstrate key roles for the cholinergic system in the mechanisms of alcoholic pancreatitis.
Pancreas; alcohol feeding; cerulein; cholinergic pathways; acetylcholinesterase
The exocrine pancreas has the greatest protein synthetic capacity of any mammalian organ and is challenged with the synthesis, processing and transporting a large load of digestive enzymes. Based on recent findings we present a hypothesis proposing that mutations in the digestive enzymes and environmental risks impacting the pancreas (i.e., alcohol abuse, smoking, metabolic disorders, and drugs) cause endoplasmic reticulum (ER) stress. We review recent findings showing that in normal pancreas the ER stress resulting from alcohol abuse leads to an adaptive unfolded protein response (UPR) allowing for maintenance of protein synthesis, processing, and transport. However, when key pathways necessary for the adaptive UPR are altered, the exocrine cell of the pancreas is unable to maintain these processes and cellular pathology results. These findings may explain why some individuals with alcohol abuse disorders develop organ injury and disease while most do not. Further, the findings allow us to hypothesize that the UPR in the exocrine pancreas adapts the protein synthetic machinery of the ER stress resulting from mutational and environmental stressors. When the ability of the UPR to adapt to the stressors is exceeded, pathologic pathways and disease develop.
UPR; pancreas; gastrointestinal; research; exocrine; pancreatic function
To define the role of protein kinase C δ (PKC δ) in acinar cell responses to the hormone cholecystokinin-8 (CCK) using isoform-specific inhibitors and a previously unreported genetic deletion model.
Pancreatic acinar cells were isolated from 1) rat, and pre-treated with a PKC-δ-specific inhibitor or 2) PKC δ deficient and wild type mice. Isolated cells were stimulated with CCK (0.001-100 nM) and cell responses measured.
The PKC δ inhibitor did not affect stimulated amylase secretion from rat pancreatic acinar cells. CCK stimulation induced a typical biphasic dose response curve for amylase secretion in acinar cells isolated from both PKC δ -/- and wild type mice, with maximal stimulation at 10 pM CCK. CCK (100 nM) induced zymogen and NFkB activation in both PKC δ -/- and wild type mice, although it was up to 50% less in PKC δ -/-.
In contrast to previous studies, this study has used specific and complimentary approaches to examine PKC δ mediated acinar cell responses. We could not confirm that it mediates amylase release, but corroborated its role in the early stages of acute pancreatitis.
Background & Aims:
The plasminogen (plg) system participates in tissue repair in several
organs, but its role in pancreas repair remains poorly characterized. To
better understand the role of plg in pancreas recovery following injury, we
examined the course of caerulein-induced pancreatitis in plg deficient and
Pancreatitis was induced by caerulein administration (50
μg/kg, 7 ip injections). Mice were sacrificed either at the acute
phase (7 hours after the first caerulein injection) or during recovery (at
2, 4 and 7 days). In pancreatic sections we examined: pancreatic morphology,
trypsin activation, inflammatory cell infiltration, acinar cell death, cell
proliferation, extracellular matrix (ECM) deposition, activation of stellate
cells (PSCs), and components of the plg and metalloproteinase systems.
In plg sufficient mice, pancreatic plg levels and plasmin activity
increased during the acute phase and remained elevated during recovery.
Pancreatitis resolved in plg sufficient mice within 7 days. Pancreas
recovery involved reorganization of the parenchyma structure, removal of
necrotic debris, cell proliferation, transient activation of PSCs and
moderate deposition of ECM proteins. Acute pancreatitis (7-h) was
indistinguishable between plg deficient and sufficient mice. In contrast,
pancreas recovery was impaired in plg deficient mice. Plg deficiency led to
disorganized parenchyma, extensive acinar cell loss, poor removal of
necrotic debris, reduced cell proliferation and fibrosis. Fibrosis was
characterized by deposition of collagens and fibronectin, persistent
activation of PSCs and upregulation of pancreatic TGF-β1.
Plg/plasmin deficiency leads to features similar to those found in
chronic pancreatitis such as parenchymal atrophy and fibrosis.
Activated stellate cells are considered the principal mediators of chronic alcoholic pancreatitis/fibrosis. However the mechanisms of alcohol action on pancreatic stellate cells (PaSCs) are poorly understood. The aims of this study were to determine the presence and role of the NADPH oxidase system in mediating alcohol effects on PaSCs with specific emphasis on proliferation.
PaSC NADPH oxidase components mRNA and protein were determined by RT-PCR and Western blot. The NADPH oxidase activity was measured by detecting the production of reactive oxygen species using lucigenin-derived chemiluminescence assay. PaSC DNA synthesis, a measure of proliferation, was performed by determining the [3H] thymidine incorporation into DNA.
mRNA for NADPH oxidase components Nox1, gp91phox, Nox4, p22phox, p47phox and p67phox and protein for NADPH oxidase subunits gp91phox, p22phox, p47phox and p67phox are present in PaSCs. Treatment with platelet-derived growth factor (PDGF) significantly increased the NADPH oxidase activity and DNA synthesis in cultured PaSCs. Alcohol treatment markedly augmented both the NADPH oxidase activity and the DNA synthesis caused by PDGF, which was prevented by antioxidant N-acetyl-L-cysteine, ROS scavenger tiron, and the NADPH oxidase inhibitor diphenylene iodium. The effects of PDGF on NADPH oxidase activity and DNA synthesis were prevented in PaSCs isolated from the pancreas of mice with a genetic deficiency of p47phox.
Ethanol causes proliferation of stellate cells by augmenting the activation of the cell's NADPH oxidase system stimulated by PDGF. These results provide new insights into the mechanisms of alcohol-induced fibrosing disorders.
NADPH oxidase; Reactive oxygen species; Proliferation; Platelet-derived growth factor; Ethanol
Pancreatic stellate cells (PaSCs) are myofibroblast-like cells found in the areas of the pancreas that have exocrine function. PaSCs are regulated by autocrine and paracrine stimuli and share many features with their hepatic counterparts, studies of which have helped further our understanding of PaSC biology. Activation of PaSCs induces them to proliferate, to migrate to sites of tissue damage, to contract and possibly phagocytose, and to synthesize ECM components to promote tissue repair. Sustained activation of PaSCs has an increasingly appreciated role in the fibrosis that is associated with chronic pancreatitis and with pancreatic cancer. Therefore, understanding the biology of PaSCs offers potential therapeutic targets for the treatment and prevention of these diseases.