Nicotine facilitates pancreatic fibrosis by promoting activation of pancreatic stellate cells via α7nAChR-mediated JAK2/STAT3 signaling pathway in rats.

AIM: Smoking has been considered as a risk factor of chronic pancreatitis (CP), but the potential mechanism is still unknown. The major pathological feature of CP is pancreatic fibrosis, whose major functional cells are pancreatic stellate cells (PSCs). Nicotine is the major component of cigarette smoke, our recent study suggested that nicotine has the potential to facilitate pancreatic fibrosis in CP. This study was aimed to analyze the function and mechanism of nicotine on PSCs and pancreatic fibrosis in rats. MATERIALS AND METHODS: In vivo, a rat CP model was induced by intraperitoneal injection of 20 % L-arginine hydrochloride (200 mg/100 g) at 1 h intervals twice per week, nicotine was injected subcutaneously at a dose of 1 mg/kg body weight per day. After four weeks, the pancreatic tissue was collected for H&E, Masson and immunohistochemical staining. In vitro, primary rPSCs were isolated from rats and treated with nicotine (0.1 µM and 1 µM). The proliferation、apoptosis、α-SMA expression、extracellular matrix (ECM) metabolism and α7nAChR-mediated JAK2/STAT3 signaling pathway of rPSCs were detected by CCK-8 assay、flow cytometry、real-time Q-PCR and western blotting analysis. The α7nAChR antagonist α-bungarotoxin (α-BTX) was used to perform inhibition experiments. KEY FINDINGS: Nicotine increased pancreatic damage, collagen deposition and activation of PSCs in the CP rat model. In rPSCs, the proliferation, α-SMA expression and ECM formation were significantly promoted by nicotine in a dose-dependent manner. Meanwhile, the apoptosis of rPSCs was significantly reduced after nicotine treatment. Moreover, nicotine also activated the α7nAChR-mediated JAK2/STAT3 signaling pathway in rPSCs. These effects of nicotine on rPSCs were blocked by α-BTX. SIGNIFICANCE: Our finding in this research suggests that nicotine facilitates pancreatic fibrosis by promoting activation of pancreatic stellate cells via α7nAChR-mediated JAK2/STAT3 signaling pathway in rats, partly revealing the mechanism of smoking on chronic pancreatitis.

Disease duration before surgical resection for chronic pancreatitis impacts long-term outcome.

Many patients with chronic pancreatitis (CP) undergo a step-up approach with interventional procedures as first-line treatment and resection reserved for later stages. The aim of this study was to identify predictive factors for a significant clinical improvement (SCI) after surgical treatment.All patients operated for CP between September 2012 and June 2017 at our center was retrospectively reviewed. A prospective patient survey was conducted to measure patients postoperative outcome. The primary endpoint SCI was defined as stable health status, positive weight development and complete pain relief without routine pain medication. Additionally, risk factors for relaparotomy were analyzed.A total of 89 patients with a median follow-up of 38 months were included. In most cases, a duodenum-preserving pancreatic head resection (n = 48) or pancreatoduodenectomy (n = 28) was performed. SCI was achieved in 65.3% (n = 47) of the patients after the final medium follow-up of 15.0 months (IQR: 7.0-35.0 months), respectively. Patients with a longer mean delay (7.7 vs 4 years) between diagnosis and surgical resection were less likely to achieve SCI (P = .02; OR .88; 95%CI .80-98). An endocrine insufficiency was a negative prognostic factor for SCI (P = .01; OR .15; 95%CI .04-68). In total, 96.2% of the patients had a complete or major postoperative relief with a mean pain intensity reduction from 8.1 to 1.9 on the visual analogue scale.The results support that surgical resection for CP should be considered at early stages. Resection can effectively reduce postoperative pain intensity and improve long-term success.

Nimbolide abrogates cerulein-induced chronic pancreatitis by modulating ß-catenin/Smad in a sirtuin-dependent way.

Chronic pancreatitis (CP) is one of the leading causes of mortality worldwide with no clinically approved therapeutic interventions. The present study was designed to investigate the protective effect of nimbolide (NB), an active constituent of neem tree (Azadirachta indica), by targeting ß-catenin/Smad/SIRT1 in cerulein-induced CP model. The effects of NB was investigated on cerulein (50 µg/kg/hr*6 exposures /day, 3 days a week for 3 weeks) induced CP in mice. Amylase and lipase activity were measured and histopathological evaluation was performed. Collagen deposition in the pancreatic tissue was estimated by hydroxyproline assay, and collagen specific staining picrosirius red and Masson's trichrome. Cerulein-induced CP was significantly controlled by NB treatment, as shown by the downregulation of ß-catenin/Smad signaling in a SIRT1 dependent manner. NB treatment significantly decreased α-SMA, MMP-2, collagen1a, fibronectin, TGF-ß1, p-Smad-2/3 expression and extracellular matrix (ECM) deposition in pancreatic tissue. However, the protective effects of NB on cerulein-induced CP were undermined by nicotinamide (NMD) or splitomicin, sirtuin 1 (SIRT1) inhibitors treatment. NB treatment modulated protein expression by activating SIRT1 and decreasing the expression of ß-catenin/Smad proteins in CP mice. However, the expression of SIRT1 in pancreatic tissue was elevated by NB treatment and it was decreased by NMD or splitomicin treatment. In summary, our results strongly suggest that NB exerted promising protective effects in cerulein-induced CP model by inhibiting ß-catenin/Smad in a sirtuin-dependent manner, which could be attributed to its anti-inflammatory and antifibrotic effects. Our study suggests that NB could be an effective therapeutic intervention for the treatment of CP.

Mutation That Promotes Activation of Trypsinogen Increases Severity of Secretagogue-Induced Pancreatitis in Mice.

BACKGROUND & AIMS: Mutations in the human serine protease 1 gene (PRSS1), which encodes cationic trypsinogen, can accelerate its autoactivation and cause hereditary or sporadic chronic pancreatitis. Disruption of the locus that encodes cationic trypsinogen in mice (T7) causes loss of expression of the protein, but only partially decreases the severity of secretagogue-induced acute pancreatitis and has no effect on chronic pancreatitis. We investigated whether trypsinogen becomes pathogenic only when its activation is promoted by mutation. METHODS: We generated mice with knock-in of the p.K24R mutation (called T7K24R mice), which is analogous to human PRSS1 mutation p.K23R. We gave T7K24R and C57BL/6N (control) mice repeated injections of cerulein to induce pancreatitis. Plasma amylase activity, pancreatic edema, and myeloperoxidase content in pancreas and lungs were quantified. We expressed mutant and full-length forms of PRSS1 in Escherichia coli and compared their autoactivation. RESULTS: The p.K24R mutation increased autoactivation of T7 5-fold. T7K24R mice developed no spontaneous pancreatitis. T7K24R mice given cerulein injections had increased pancreatic activation of trypsinogen and more edema, infiltration of lung and pancreas by inflammatory cells, and plasma amylase activity compared with control mice given cerulein injections. Injection of cerulein for 2 days induced progressive pancreatitis in T7K24R mice, but not in control mice, with typical features of chronic pancreatitis. CONCLUSIONS: Introduction of a mutation into mice that is analogous to the p.K23R mutation in PRSS1 increases pancreatic activation of trypsinogen during secretagogue-induced pancreatitis. Higher pancreatic activity of trypsin increases the severity of pancreatitis, even though loss of trypsin activity does not prevent pancreatitis in mice.

Pancreatic pleural effusion masquerading as right sided tubercular pleural effusion.

Pleural effusion is easily diagnosed often managed optimally with standard protocols. It at times, is a diagnostic dilemma as it comes with big list of differential diagnosis. Pleural effusion due to pancreaticopleural fistula (PPF) is a rare and on right side is even rarer. Detailed history along with high index of suspicion in required to diagnose PPF, which is confirmed by increased level of pleural fluid amylase and lipase along with magnetic resonance cholangiopancreatography (MRCP) or endoscopic retrograde cholangiopancreatography (ERCP) demonstrating fistula tract. Here we report the case of a young patient who presented with respiratory distress and was wrongly diagnosed as right sided tubercular effusion which later turned out to be pancreatic effusion. Management in our case was multi-disciplinary involving pulmonologist, gastroenterologist, radiologist and thoracic surgeon.

Pancreas-specific plasma amylase for assessment and diagnosis of chronic pancreatitis: New insights on an old topic.

Background: Pancreatic function testing and imaging are used to inform the diagnosis of chronic pancreatitis, but most of these methods are time- and cost-consuming or lack diagnostic accuracy. Objective: We investigated the utility of pancreas-specific plasma amylase for assessment and diagnosis of chronic pancreatitis. Design: This was a prospective study of 121 consecutive patients with chronic pancreatitis and a reference population of 94 healthy controls. Pancreas-specific plasma amylase level was determined and analysed for its association with exocrine pancreatic insufficiency, diabetes and other clinical variables. Receiver operating characteristic curve analyses were performed to determine the diagnostic utility of plasma amylase for diagnosing chronic pancreatitis and to study associations with disease severity. The findings were validated in a further cohort of 57 patients with chronic pancreatitis. Results: Significant and independent associations between plasma amylase level and duration of chronic pancreatitis as well as the presence of exocrine pancreatic insufficiency and diabetes were observed (all p < 0.001). An amylase level below 17.3 U/l had a high specificity (94%) and moderate sensitivity (59%) for the diagnosis of chronic pancreatitis. Diagnostic performance was influenced by disease stage with the best performance observed for advanced disease. The findings were replicated in the validation cohort. Conclusion: Pancreas-specific plasma amylase may provide a clinically useful mean for assessment and diagnosis of chronic pancreatitis.

Chronic Asymptomatic Pancreatic Hyperenzymemia: A Long-term Follow-up.

OBJECTIVES: Chronic asymptomatic pancreatic hyperenzymemia (CAPH) was described as a benign disease. However, we already described clinically relevant findings requiring surgery or follow-up in half of the subjects. The aim of this study was to evaluate the long-term outcome of CAPH in terms of symptoms and evolution toward chronic pancreatitis. METHODS: Subjects previously enrolled in the first phase of the study (from 2005 to 2010) were reinvestigated from December 2013 to January 2017 with a phone call ± magnetic resonance cholangiopancreatography with secretin stimulation. RESULTS: A total of 133 subjects were eligible for the follow-up study (75 males, 58 females; age, 48.4 [standard deviation {SD}, 14] years); 24 (18%) of them dropped out. During a mean follow-up of 9.3 (SD, 5.2) years after the first diagnosis of CAPH, no episode of acute pancreatitis or abdominal pain was reported. Sixty-three subjects (58%) of 109 underwent magnetic resonance cholangiopancreatography with secretin stimulation with a mean follow-up of 5.7 [SD, 3.1] years (range, 1-11 years). Secretin stimulation-MRCP resulted unchanged in 54 (90%) of 60 subjects, worsened in 3 (5%) and improved in 3 (5%). Two subjects died from causes unrelated to pancreatic disease. CONCLUSIONS: Excluding subjects with a pancreatic disease at index magnetic resonance imaging, CAPH is a benign condition.

Human CPA1 mutation causes digestive enzyme misfolding and chronic pancreatitis in mice.

OBJECTIVE: Chronic pancreatitis is a progressive, relapsing inflammatory disorder of the pancreas, which often develops in the background of genetic susceptibility. Recently, loss-of-function mutations in CPA1, which encodes the digestive enzyme carboxypeptidase A1, were described in sporadic early onset cases and in hereditary pancreatitis. Mutation-induced misfolding of CPA1 and associated endoplasmic reticulum (ER) stress was suggested as potential disease mechanism; however, in vivo evidence has been lacking. The objective of the present study was to create a mouse model that recapitulates features of CPA1-associated chronic pancreatitis. DESIGN: We knocked-in the most frequently occurring p.N256K human CPA1 mutation to the mouse Cpa1 locus. Mutant mice were characterised with respect to pancreas pathology and ER stress and compared with C57BL/6N and CPA1 null control mice. RESULTS: In the CPA1 N256K mutant mice, we observed hallmarks of chronic pancreatitis that included progressive acinar cell atrophy, inflammatory cell infiltration, fibrosis and acinar-ductal metaplasia. In contrast, similarly to the C57BL/6N mice, the CPA1 null control strain exhibited no signs of pancreatic disease. Mutation p.N256K induced misfolding of mouse CPA1 and resulted in elevated expression of ER stress markers Hspa5 (BiP) and Ddit3 (CHOP) both in cell culture and mutant mice. CONCLUSION: The results offer categorical evidence that CPA1 mutations elicit enzyme misfolding and cause chronic pancreatitis via an ER stress-related mechanism.

The effect of primary hyperparathyroidism on pancreatic exocrine function.

BACKGROUND: Elastase-1 is a proteolytic enzyme secreted by pancreatic acinar cells, and measurements of the concentration this enzyme are used to evaluate pancreatic exocrine function. We aimed to determine whether pancreatic exocrine function declines due to chronic hypercalcemia by measuring fecal elastase levels. METHODS: 75 patients with primary hyperparathyroidism (18 men and 47 women) and 30 healthy subjects (11 men and 19 women) participated in this study. Renal function tests, lipid parameters, bone mineral density, and serum calcium, phosphorus, vitamin D, parathormone, glucose, and thyroid stimulating hormone levels as well as fecal elastase concentrations, were determined in these patients and controls. RESULTS: The mean fecal elastase level was 335.3 ± 181.4 µg/g in the PHPT group and 317.4 ± 157.3 µg/g in the control group. There was no significant difference in fecal elastase levels between the two groups (p = 0.5). CONCLUSIONS: Chronic hypercalcemia in primary hyperparathyroidism did not decrease the fecal elastase level, which is an indirect indicator of chronic pancreatitis; therefore, chronic hypercalcemia in PHPT may not cause chronic pancreatitis.

No significant enrichment of rare functionally defective CPA1 variants in a large Chinese idiopathic chronic pancreatitis cohort.

Rare functionally defective carboxypeptidase A1 (CPA1) variants have been reported to predispose to nonalcoholic chronic pancreatitis, mainly the idiopathic subtype. However, independent replication has so far been lacking, particularly in Asian cohorts where initial studies employed small sample sizes. Herein we performed targeted next-generation sequencing of the CPA1 gene in 1,112 Han Chinese idiopathic chronic pancreatitis (ICP) patients-the largest ICP cohort so far analyzed in a single population-and 1,580 controls. Sanger sequencing was used to validate called variants, and the CPA1 activity and secretion of all newly found variants were measured. A total of 18 rare CPA1 variants were characterized, 11 of which have not been previously described. However, no significant association was noted with ICP irrespective of whether all rare variants [20 out of 1,112 (1.8%) in patients vs. 24 out of 1,580 (1.52%) in controls; P = 0.57] or functionally impaired variants [three out of 1,112 (0.27%) in patients vs. two out of 1,580 (0.13%) in controls; P = 0.68] were considered.

Endoscopic Ultrasonography May Select Subjects Having Asymptomatic Chronic Pancreatic Hyperenzymemia Who Require a Stricter Follow-up.

OBJECTIVES: We have previously shown that at least 50% of patients with asymptomatic chronic pancreatic hyperenzymemia (ACPH) may develop morphological pancreatic alterations. Endoscopic ultrasonography (EUS) may detect small lesions, and its sensitivity seems to be higher than other imaging techniques. The aim of this study was to evaluate whether EUS may modify the management of patients having ACPH. METHODS: In 2 referral centers for pancreatic disease, a retrospective analysis of prospectively enrolled patients with ACPH was conducted. RESULTS: Seventy-three patients with ACPH were enrolled for the purpose of this study. Endoscopic ultrasonography was performed as the last examination in 45 subjects who resulted negative at previous imaging studies (abdominal ultrasound, computed tomography, magnetic resonance imaging associated with cholangiopancreatography). Using EUS in 7 subjects, abnormalities were found in the following: 3 branch-duct intraductal papillary mucinous neoplasms, 1 duodenal diverticulum, 1 annular pancreas, 1 findings suggestive of chronic pancreatitis, and 1 undefined cyst (<5 mm). CONCLUSIONS: Endoscopic ultrasonography is able to detect alteration not found by other imaging technique in 15.5% of patients with ACPH and may be useful to select those patients who require a more strict follow-up.

Nationwide epidemiological survey of early chronic pancreatitis in Japan.

BACKGROUND: The world's first diagnostic criteria for early CP were proposed in 2009 in Japan. This study aimed to clarify the clinico-epidemiological features of early CP in Japan. METHODS: Patients with early CP who were diagnosed according to the diagnostic criteria for early CP and had visited the selected hospitals in 2011 were surveyed. The study consisted of two-stage surveys: the number of patients with early CP was estimated by the first questionnaire and their clinical features were assessed by the second questionnaire. RESULTS: The estimated number of early CP patients was 5410 (95% confidence interval 3675-6945), with an overall prevalence of 4.2 per 100,000 persons. The number of patients who were newly diagnosed with early CP was estimated to be 1330 (95% confidence interval 1058-1602), with an annual incidence of 1.0 per 100,000 persons. Detailed clinical information was obtained in 151 patients in the second survey. The male-to-female sex ratio was 1.32:1. The mean age was 60.4 and the mean age at disease onset was 55.4. Idiopathic (47.7%) and alcoholic (45.0%) were the two most common etiologies. Proportions of female and idiopathic cases were higher in early CP than in definite CP. Hyperechoic foci without shadowing and stranding were the most common findings on endoscopic ultrasonography. The clinical profiles of early CP patients who showed lobularity with honeycombing on endoscopic ultrasonography or previous episodes of acute pancreatitis were similar to those of definite CP patients. CONCLUSIONS: We clarified the current status of early CP in Japan.

The MET Receptor Tyrosine Kinase Confers Repair of Murine Pancreatic Acinar Cells following Acute and Chronic Injury.

Acinar cells represent the primary target in necroinflammatory diseases of the pancreas, including pancreatitis. The signaling pathways guiding acinar cell repair and regeneration following injury remain poorly understood. The purpose of this study was to determine the importance of Hepatocyte Growth Factor Receptor/MET signaling as an intrinsic repair mechanism for acinar cells following acute damage and chronic alcohol-associated injury. Here, we generated mice with targeted deletion of MET in adult acinar cells (MET-/-). Acute and repetitive pancreatic injury was induced in MET-/- and control mice with cerulein, and chronic injury by feeding mice Lieber-DeCarli diets containing alcohol with or without enhancement of repetitive pancreatic injury. We examined the exocrine pancreas of these mice histologically for acinar death, edema, inflammation and collagen deposition and changes in the transcriptional program. We show that MET expression is relatively low in normal adult pancreas. However, MET levels were elevated in ductal and acinar cells in human pancreatitis specimens, consistent with a role for MET in an adaptive repair mechanism. We report that genetic deletion of MET in adult murine acinar cells was linked to increased acinar cell death, chronic inflammation and delayed recovery (regeneration) of pancreatic exocrine tissue. Notably, increased pancreatic collagen deposition was detected in MET knockout mice following repetitive injury as well alcohol-associated injury. Finally, we identified specific alterations of the pancreatic transcriptome associated with MET signaling during injury, involved in tissue repair, inflammation and endoplasmic reticulum stress. Together, these data demonstrate the importance of MET signaling for acinar repair and regeneration, a novel finding that could attenuate the symptomology of pancreatic injury.

A Carboxyl Ester Lipase (CEL) Mutant Causes Chronic Pancreatitis by Forming Intracellular Aggregates That Activate Apoptosis.

Patients with chronic pancreatitis (CP) frequently have genetic risk factors for disease. Many of the identified genes have been connected to trypsinogen activation or trypsin inactivation. The description of CP in patients with mutations in the variable number of tandem repeat (VNTR) domain of carboxyl ester lipase (CEL) presents an opportunity to study the pathogenesis of CP independently of trypsin pathways. We tested the hypothesis that a deletion and frameshift mutation (C563fsX673) in the CEL VNTR causes CP through proteotoxic gain-of-function activation of maladaptive cell signaling pathways including cell death pathways. HEK293 or AR42J cells were transfected with constructs expressing CEL with 14 repeats in the VNTR (CEL14R) or C563fsX673 CEL (CEL maturity onset diabetes of youth with a deletion mutation in the VNTR (MODY)). In both cell types, CEL MODY formed intracellular aggregates. Secretion of CEL MODY was decreased compared with that of CEL14R. Expression of CEL MODY increased endoplasmic reticulum stress, activated the unfolded protein response, and caused cell death by apoptosis. Our results demonstrate that disorders of protein homeostasis can lead to CP and suggest that novel therapies to decrease the intracellular accumulation of misfolded protein may be successful in some patients with CP.

Telomerase activity in pancreatic juice differentiates pancreatic cancer from chronic pancreatitis: A meta-analysis.

BACKGROUND/OBJECTIVE: To evaluate the usefulness of genetic markers in pancreatic juice (PJ), and the combination of these markers with telomerase activity in the differential diagnosis of pancreatic ductal adenocarcinoma (PDAC) from chronic pancreatitis. METHODS: We conducted a meta-analysis for the diagnostic utility of the four major altered genes in PDAC (KRAS, CDKN2A/p16, TP53, and SMAD4/DPC4), telomerase activity, and a combination assay using PJ samples. A literature search was conducted in MEDLINE, Cochrane Library, and Web of Science. Data were pooled and presented as diagnostic sensitivity and specificity with 95% confidence intervals (CIs). RESULTS: Thirty-nine studies fulfilled the inclusion criteria. Pooled estimates of KRAS analysis were as follows: sensitivity was 0.67 (95% CI, 0.63-0.71) and specificity, 0.82 (95% CI, 0.79-0.85). For telomerase activity analysis, sensitivity was 0.82 (95% CI, 0.76-0.87) and specificity, 0.96 (95% CI, 0.90-0.99). The other three tumor suppressors demonstrated low sensitivity. The data did not suggest any publication bias. A combined analysis of KRAS and telomerase activity showed a higher diagnostic sensitivity (0.94; 95% CI, 0.83-0.99) than KRAS alone. A combined analysis of telomerase activity and cytology revealed more reliable diagnostic accuracy than telomerase activity alone, with high sensitivity (0.88; 95% CI, 0.74-0.96) and specificity (1.00; 95% CI, 0.91-1.00). CONCLUSIONS: The most reliable marker in PJ samples for diagnosis of PDAC was telomerase activity. Telomerase activity can play a central role in diagnostic analysis using PJ samples, and can increase diagnostic accuracy when combined with KRAS mutations or cytological examination.

Pathogenic cellular role of the p.L104P human cationic trypsinogen variant in chronic pancreatitis.

Mutations in the PRSS1 gene encoding human cationic trypsinogen are associated with hereditary and sporadic chronic pancreatitis. High-penetrance PRSS1 mutations found in hereditary pancreatitis alter activation and/or degradation of cationic trypsinogen, thereby promoting intrapancreatic trypsinogen activation. In contrast, a number of rare PRSS1 variants identified in subjects with sporadic chronic pancreatitis cause misfolding and endoplasmic reticulum (ER) stress. Mutation p.L104P is unique among natural PRSS1 variants, since it affects the substrate binding site of trypsin. The aim of the present study was to establish the clinical significance of variant p.L104P through functional analysis. We found that p.L104P trypsin exhibited decreased activity on peptide and protein substrates; however, autoactivation was slightly accelerated. Remarkably, binding of the physiological trypsin inhibitor serine protease inhibitor Kazal type 1 (SPINK1) was decreased by 70-fold. In the presence of the trypsinogen-degrading enzyme chymotrypsin C, mutant p.L104P autoactivated to higher trypsin levels than wild-type trypsinogen. This apparent resistance to degradation was due to slower cleavage at Arg(122) rather than Leu(81) Finally, secretion of mutant p.L104P from transfected cells was markedly reduced due to intracellular retention and aggregation with concomitant elevation of ER stress markers. We conclude that PRSS1 variant p.L104P exhibits a variety of phenotypic changes that can increase risk for chronic pancreatitis. Mutation-induced misfolding and associated ER stress are the dominant effects that support a direct pathogenic role in chronic pancreatitis.

Inhibition of Chronic Pancreatitis and Murine Pancreatic Intraepithelial Neoplasia by a Dual Inhibitor of c-RAF and Soluble Epoxide Hydrolase in LSL-KrasG¹²D/Pdx-1-Cre Mice.

Mutation of Kirsten rat sarcoma viral oncogene homolog (KRAS) and chronic pancreatitis are the most common pathogenic events involved in human pancreatic carcinogenesis. In the process of long-standing chronic inflammation, aberrant metabolites of arachidonic acid play a crucial role in promoting carcinogenesis, in which the soluble epoxide hydrolase (sEH), as a pro-inflammatory enzyme, generally inactivates anti-inflammatory epoxyeicosatrienoic acids (EETs). Herein, we determined the effect of our newly-synthesized novel compound trans-4-{4-[3-(4-chloro-3-trifluoromethyl-phenyl)-ureido]-cyclohexyloxy}-pyridine-2-carboxylic acid methylamide (t-CUPM), a dual inhibitor of sEH and RAF1 proto-oncogene serine/threonine kinase (c-RAF), on inhibiting the development of pancreatitis and pancreatic intraepithelial neoplasia (mPanIN) in LSL-Kras(G12D)/Pdx1-Cre mice. The results showed that t-CUPM significantly reduced the severity of chronic pancreatitis, as measured by the extent of acini loss, inflammatory cell infiltration and stromal fibrosis. The progression of low-grade mPanIN I to high-grade mPanIN II/III was significantly suppressed. Inhibition of mutant Kras-transmitted phosphorylation of mitogen-activated protein kinase's kinase/extracellular signal-regulated kinases was demonstrated in pancreatic tissues by western blots. Quantitative real-time polymerase chain reaction analysis revealed that t-CUPM treatment significantly reduced the levels of inflammatory cytokines including tumor necrosis facor-α, monocyte chemoattractant protein-1, as well as vascular adhesion molecule-1, and the levels of Sonic hedgehog and Gli transcription factor (Hedgehog pathway). Analysis of the eicosanoid profile revealed a significant increase of the EETs/dihydroxyeicosatrienoic acids ratio, which further confirmed sEH inhibition by t-CUPM. These results indicate that simultaneous inhibition of sEH and c-RAF by t-CUPM is important in preventing chronic pancreatitis and carcinogenesis.

Phosphatidylinositol 3-Kinase: A Link Between Inflammation and Pancreatic Cancer.

Even though a strong association between inflammation and cancer has been widely accepted, the underlying precise molecular mechanisms are still largely unknown. A complex signaling network between tumor and stromal cells is responsible for the infiltration of inflammatory cells into the cancer microenvironment. Tumor stromal cells such as pancreatic stellate cells (PSCs) and immune cells create a microenvironment that protects cancer cells through a complex interaction, ultimately facilitating their local proliferation and their migration to different sites. Furthermore, PSCs have multiple functions related to local immunity, angiogenesis, inflammation, and fibrosis. Recently, many studies have shown that members of the phosphoinositol-3-phosphate kinase (PI3K) family are activated in tumor cells, PSCs, and tumor-infiltrating inflammatory cells to promote cancer growth. Proinflammatory cytokines and chemokines secreted by immune cells and fibroblasts within the tumor environment can activate the PI3K pathway both in cancer and inflammatory cells. In this review, we focus on the central role of the PI3K pathway in regulating the cross talk between immune/stromal cells and cancer cells. Understanding the role of the PI3K pathway in the development of chronic pancreatitis and cancer is crucial for the discovery of novel and efficacious treatment options.

Trametinib and dactolisib but not regorafenib exert antiproliferative effects on rat pancreatic stellate cells.

BACKGROUND: Modulation of the stroma response is considered a promising approach for the treatment of chronic pancreatitis and pancreatic cancer. The aim of this study was to evaluate the effects of three clinically available small molecule kinase inhibitors, regorafenib, trametinib and dactolisib, on effector functions of activated pancreatic stellate cells (PSCs), which play a key role in pancreatic fibrosis. METHODS: Cultured rat PSCs were exposed to small molecule kinase inhibitors. Proliferation and cell death were assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine and cytotoxicity, respectively. Levels of mRNA were determined by real-time PCR, while protein expression and phosphorylation were analyzed by immunoblotting. Interleukin-6 levels in culture supernatants were quantified by ELISA. Zymography assays were performed to monitor collagenase activity in culture supernatants. RESULTS: The MEK inhibitor trametinib and the dual phosphatidylinositol 3-kinase/mTOR inhibitor dactolisib, but not the multi-kinase inhibitor regorafenib, efficiently inhibited PSC proliferation. Trametinib as well as regorafenib suppressed the expression of two autocrine mediators of PSC activation, interleukin-6 and transforming growth factor-beta1. Dactolisib-treated cells expressed less alpha1 type I collagen and lower levels of alpha-smooth muscle actin, a marker of the myofibroblastic PSC phenotype. Simultaneous application of dactolisib and trametinib displayed additive inhibitory effects on cell growth without statistically significant cytotoxicity. Activity of matrix metalloproteinase-2 was not affected by any of the drugs. CONCLUSION: We suggest the combination of two drugs, that specifically target two key signaling pathways in PSC, Ras-Raf-MEK-ERK (trametinib) and phosphatidylinositol 3-kinase-AKT-mTOR (dactolisib), as a concept to modulate the activation state of the cells in the context of fibrosis.