Introduction

Pancreatic cancer is particularly notable for its aggressive progression and dismal prognosis, which underscores the urgent need for the development of novel therapeutic strategies that can effectively combat this formidable disease. Recent investigations have brought to light the potential of salvianolic acid B (SalB), a polyphenolic compound that is extracted from the roots of the Salvia miltiorrhiza plant, as a promising candidate for targeted cancer treatment. However, the precise mechanisms through which SalB influences metabolic changes in pancreatic cancer cells and promotes ferroptosis—a specific type of programmed cell death characterized by iron-dependent lipid peroxidation—remain to be fully elucidated. Therefore, this study aims to thoroughly investigate the impact of SalB on the metabolic reprogramming of pancreatic cancer cells, while also clarifying the specific mechanisms by which it induces ferroptosis.

Aims & Methods

We established an in situ mouse model of pancreatic cancer to evaluate SalB's anti-tumor effects at 10, 30, and 100 μM, assessing critical parameters like glutathione (GSH) levels, glutathione peroxidase 4 (GPX4) activity, malondialdehyde (MDA) production, reactive oxygen species (ROS) levels, ferrous iron (Fe2+) concentrations, mitochondrial morphology, ATP levels, enzyme activity, mitochondrial ROS, and mitochondrial membrane potential (MMP) in Panc-1, BxPC-3, and SW1990 cell lines. We also used Fer-1 to inhibit ferroptosis and reevaluated SalB's effects, exploring its relationship with ferroptosis in pancreatic cancer cells.

Results

SalB significantly reduced tumor volume in mice afflicted with in situ pancreatic cancer, all while notably not impacting the body weight of these animals, suggesting a targeted therapeutic effect without the common side effects associated with weight loss. In various cell lines, including Panc-1, BxPC-3, and SW1990, treatment with SalB resulted in a marked decline in levels of GSH and GPX4, which are crucial for cellular antioxidant defense. This decline was coupled with a significant increase in MDA and ROS, indicating that SalB effectively induces oxidative stress within the cells, a condition that can lead to cell death. Furthermore, SalB treatment led to elevated intracellular levels of Fe2+, alterations in mitochondrial morphology, a decrease in MMP, and reduced ATP levels, alongside inhibited activity of mitochondrial respiratory chain enzymes and increased production of mitochondrial ROS. These changes collectively confirm SalB's potent ability to inhibit mitochondrial function while simultaneously improving the overall metabolic condition in pancreatic cancer cells. Following pretreatment with the ferroptosis inhibitor Fer-1, the reductions in GSH, GPX4, and MMP levels, as well as the increases in MDA, ROS, and Fe2+ levels induced by SalB, were effectively reversed, demonstrating the dynamic interplay between these cellular components. Additionally, Fer-1 restored ATP levels and the diminished activity of mitochondrial respiratory chain enzymes caused by SalB treatment, while also attenuating the production of mitochondrial ROS. These findings underscore the critical role of ferroptosis in mediating the effects of SalB on mitochondrial function and highlight its significant impact on the overall metabolic state of pancreatic cancer cells, suggesting a promising avenue for therapeutic intervention.

Conclusion

SalB effectively suppresses pancreatic cancer progression by inducing ferroptosis and mitochondrial metabolic reprogramming.

References

[1] Riva G, Pea A, Pilati C, et al. Histo-molecular oncogenesis of pancreatic cancer: From precancerous lesions to invasive ductal adenocarcinoma. World J Gastrointest Oncol. 2018;10(10):317-327.
[2] Li M, Tao J, Qian R, et al. Development of alternative herbals remedy for gastric cancer based on transcriptomic analysis of immune infiltration and ferroptosis. Front Genet. 14:1086368.
[3] Qin T, Rasul A, Sarfraz A, et al. Salvianolic acid A & B: potential cytotoxic polyphenols in battle against cancer via targeting multiple signaling pathways. Int J Biol Sci. 2019;15(10):2256-2264.

Introduction

Pancreatic inflammation is often triggered by sustained intraductal pressure, commonly due to gallstones or endoscopic retrograde cholangiopancreatography (ERCP). In mouse models, transient pressure disrupts tight junction integrity via calcineurin. Another key regulator of pressure-induced epithelial responses is the mechanosensitive Ca²⁺ channel PIEZO1, which detects cell elongation, increases Ca²⁺ influx, and regulates cell proliferation and apoptosis. However, studying these processes in vitro remains challenging, limiting the development of targeted therapies for post-ERCP and gallstone-induced pancreatitis.

Aims & Methods

To overcome these limitations, we aimed to utilize apical-out pancreatic organoids (hPOCs) to model pressure-related disease mechanisms ex vivo.
To investigate this we generated hPOCs from pancreatic tissue of three cadaver donors. Polarity switching was induced by Matrigel removal. Conventional and apical-out hPOCs were analyzed by RNA sequencing and live-cell fluorescent imaging. Gene expression changes in response to 1 µM PIEZO1 activator (YODA1) were assessed by qRT-PCR.

Results

Polarity switching led to reduced PIEZO1 expression and decreased intraluminal pressure, resulting in downregulation of cytoskeletal and cell cycle-related genes (ACTB, VCL, LPXN, LMNA, cyclins) as well as pro-inflammatory cytokines (CXCL1-3, CXCL8).
Heatmap analysis of transcriptomic data revealed distinct gene expression patterns between apical-in and apical-out organoids. Apical-in organoids exhibited upregulation of cell cycle and cytoskeletal genes, suggesting increased proliferation, while apical-out organoids showed a quiescent phenotype with reduced proliferative activity.
Gene Ontology (GO) analysis confirmed that apical-in organoids were enriched in pathways related to chromosome segregation, mitotic progression, and microtubule organization, whereas apical-out organoids had lower expression of these genes.
Volcano plot analysis of differentially expressed genes identified significant upregulation of MKI67, CENPF, ASPM, and UHRF1 in apical-in organoids, indicative of a proliferation-driven state. Conversely, apical-out organoids showed enrichment in DUOXA2, NUPR1, VNN2, and GBP2, genes associated with oxidative stress and inflammatory response, supporting the hypothesis that apical-in organoids reflect a pressure-induced, inflammation-like state.
Furthermore, polarity switching resulted in enhanced Cl⁻ efflux via CFTR and ANO1, likely due to the absence of sustained intraluminal pressure. Pharmacological activation of PIEZO1 by YODA1 reversed the downregulation of pro-inflammatory cytokines in apical-out hPOCs, suggesting that PIEZO1 mediates the inflammatory phenotype observed in conventional organoids.

Conclusion

Our results suggest that conventional apical-in pancreatic organoids mimic a pressure-induced inflammatory state, driven by PIEZO1-dependent signaling. In contrast, apical-out organoids, which lack sustained luminal pressure, provide a more physiological model for studying epithelial homeostasis. This platform offers new opportunities for investigating pressure-induced pancreatitis and developing targeted therapeutic strategies.

Introduction

The Revision of the Atlanta Classification (RAC) defines severe acute pancreatitis (AP) by the presence of persistent organ failure (OF), limited to respiratory, renal, or cardiovascular systems, this being associated with a high mortality rate. Whether gastrointestinal bleeding (GIB) and impaired mental status (IMS) should be considered as OF remains unclear. We aimed to assess the association of GIB and IMS with mortality and other outcomes.

Aims & Methods

We performed a retrospective analysis of a prospective database including 1,612 AP patients. GIB was defined as bleeding >500 mL. IMS was defined as a Glasgow score ≤14 at baseline. Multivariate logistic regression models were used to assess associations with mortality, controlling for comorbidity and RAC- defined OF. Two models were used: one including all OF (any duration), and another including only persistent OF.

Results

GIB occurred in 1.5% and IMS in 3% of patients. Both were associated with worse outcomes in bivariate analysis. In multivariate models, GIB was not independently associated with mortality. IMS was independently associated with mortality (aOR 4.0, 95% CI 1.2–13.4) only when adjusting for persistent OF, but not when adjusting for OF of any duration. Renal, respiratory or cardiovascular OF of any duration, as well as persistent OF, had an independent and very strong association with all the outcome variables including mortality, clearly differentiating them from GIB and IMS.

Conclusion

GIB and IMS are markers of disease severity but do not show a consistent independent association with mortality comparable to the current RAC definition of OF.

Introduction

Acute Pancreatitis (AP) is a common gastrointestinal disease with an incidence of 34 per 100,000 and mortality of up to 30% in its severe form. Frequently, AP is complicated locally and systemically. Splanchnic Venous Thrombosis (SVT) is a well-known local vascular complication of AP involving the portal vein (PV), the splenic vein (SV), and the superior mesenteric vein (SMV) in combination or alone.

Aims & Methods

The aim is to study and analyze pancreatitis-related SVT patients' epidemiological data and characteristics at the Institute of Pancreatic Diseases, Semmelweis University.From the Institute of Pancreatic Diseases, out of a total of 1141 patients hospitalized with AP between 1st January 2022 and 21st February 2024, we found 45 patients' data with pancreatitis-related thrombosis using the international code for diseases. We extracted the individual data of 45 patients from E-Med Solution and EESZT and further analyzed it. 19 patients were excluded based on inclusion and exclusion criteria.

Results

From the total of 1,141 patients, 26 were with pancreatitis-related SVT. Among these, 19 (73%) were male and 7 (27%) were female. Regarding the severity, 19 patients experienced moderate acute pancreatitis (AP), while 7 had severe AP. The most common etiologies were alcohol consumption (41%), biliary (14%), and hypertriglyceridemia (14%). The average age of the patient population was 48.3 years (SD 13.5), with male patients averaging 44.7 years (SD 10.2) and female patients averaging 58 years (SD 16.2). The average length of hospitalization was 14.5 days (SD10.4). The average BMI of the patient population was 28.4 kg/m2 (SD 4.8). Of 7 severe AP patients, 6 were males with alcoholic etiology, an average BMI of 28.9 kg/m2 (SD 3.2) and higher on admission mean Procalcitonin of 1.1 ng/mL(SD 1.23) (Vs 0.81ng/mL (SD 2.08)).
The splenic vein (SpV) was the most affected vessel, involved in 11 patients (42%), followed by superior mesenteric vein (SMV) in 3 patients (11%) and portal vein (PV) in 2 patients (7%) and 10 patients (38%) with two or more vessels involved of SpV, SMV, and PV. Of the 26 patients, 17 (65%) showed recanalization after receiving LMWH (57%) and DOAC (68%) therapy after discharge. The splenic vein recanalized the highest (68%) among the vessels.

Conclusion

In our analysis, splenic vein is the most involved vessel among the splanchnic veins and also the vessel with highest recanalization after anticoagulant therapy. Our data clearly shows anticoagulant therapy has a better outcome in patients with acute pancreatitis-related SVT. Therefore, dynamic changes of SVT should be closely monitored, and antithrombotic therapy should be rapidly initiated to prevent the further extension of thrombosis and subsequent complications. Unfortunately, major guidelines on the management of AP are silent on this aspect.Hence further studies are necessary to improve the standard of care for the AP patients with SVT.

Introduction

Alcohol is a common cause of acute pancreatitis, and ongoing consumption increases the risk of recurrent episodes. There are no international guidelines to direct behavioural or pharmacological interventions to reduce recurrence of alcohol-associated acute pancreatitis (AAP). Studies exploring the role of addiction medicine in patients with AAP are limited.

Aims & Methods

This study describes the uptake and role of the Addiction Medicine Unit (AMU), a specialist consultative service, in patients admitted with AAP. We conducted a retrospective cohort study including all inpatients diagnosed with AAP at a large multisite tertiary health service in Melbourne, Australia between May 2022 to May 2024. Patient demographics, diagnosis of alcohol use disorder (AUD) and pharmacological and non-pharmacological interventions provided by the AMU were obtained from the patient’s medical records.

Results

A total of 231 patients with 314 admissions with AAP were identified. Most were males (77.9%) with a median age of 44 years (IQR 21), with median bedside index of severity in acute pancreatitis (BISAP) score of 1 (IQR 1). Of the patients, 35 (11.1%) had necrotising pancreatitis, 30 (9.6%) required admission to the intensive care unit (ICU), and 1 patient died from AAP. The prevalence of AUD was 82.3%. Referral to the AMU occurred in 56.7% of admissions, predominantly among those with AUD (87.6%). Patients referred to the AMU had a higher median number of standard drinks per week (70 standards, range 6-385 standards) compared to those who were not referred (49 standards, range 1-301 standards) (p=0.024). The AMU recommended outpatient services alone in 40.4%, both pharmacological and outpatient interventions in 37.6%, and medications alone in 21.9%. Outpatient services included either a service for community-based assessment and counselling (86.4%), residential rehabilitation programs and/or detox services (5.8%), follow up in the AMU outpatient clinic (3.9%) or a combination of these outpatient services (3.9%). There were 137 (43.6%) admissions in which patients were commenced on medications for AUD. Topiramate (42.6%), and naltrexone (42.6%) were the most prescribed pharmacological agents by the AMU, followed by baclofen (9.8%), acamprosate (4.1%) and disulfiram (0.8%). Over the 2-year period, 54 (23.4%) were readmitted with AAP. Most patients were not readmitted within 30 days of discharge (73.2%) however there were 33 (10.5%) patients readmitted for pancreatitis or pancreatitis-related complications including pain or infection, and 18 (5.7%) readmitted to hospital for non-pancreatitis related reasons. Patients who were older (median 49 years) were more likely to be readmitted with AAP compared to those who were younger (median age 43 years) across the study period (p=0.05). There were six patients re-admitted with AAP with their index admission deemed related to an alcoholic binge without suspected underlying AUD. Of these six patients, only two were referred and reviewed by the AMU, with the other four patients not referred.

Conclusion

Our study highlights the critical role of the AMU in managing AAP, particularly given the high prevalence of concurrent AUD in this population. Despite this, significant gaps remain in screening for AUD and initiating referrals, resulting in only half of AAP patients being referred. Considering the substantial morbidity and healthcare cost associated with alcohol, efforts to improve referral practices during index admissions may be key to reduce readmissions for AAP and improve patient outcomes.

Introduction

Although we previously demonstrated that AAV8-hSPINK1 safely exhibited significant alleviation of chronic pancreatitis (CP) progression¹, the limited pancreas specificity of AAV8 leads to off-target expression of the transgenes. Additionally, the CMV promoter, as a ubiquitous promoter, lacks tissue preference, hindering its usage in AAV-mediated gene therapy for pancreatic diseases.

Aims & Methods

We aimed to construct a novel pancreas acini-specific promoter to restrict the ectopic expression of transgenes and further enhancing the precision of gene therapy for pancreatic diseases. By integrating human scRNA-seq database, the TiGER database, and our mouse RNA-seq data from Part II, we identified highly conserved genes that are richly expressed in pancreatic acini across species. Sequences surrounding the transcriptional start site (TSS, designated as +1) were selected as candidate compacted pancreas-specific promoters. We first validated transcriptional activity of the candidate promoters using dual-luciferase reporter assay. Subsequently, we packaged compacted promoters into AAV to drive ZsGreen gene expression and injected into mice. Pancreas and liver were collected and immunofluorescence, qRT-PCR, and Western blot were performed to characterised the efficiency and tissue selectivity of the candidate promoters. Finally, the synergistic effects of combining short enhancer fragments from human or rat amylase genes with the candidate promoters were assessed to enhance transcriptional activity.

Results

We selected the promoter sequences originated from hPRSS1 (408 bp), rCela1(-500/+8) (508 bp), rCela1(-205/+8) (213 bp), hSPINK1 (120 bp) as candidates. In vitro dual luciferase activity assays results indicated that the relative luciferase activities of hPRSS1 promoter and rCela1(-500/+8) promoter were 2.77±1.64 and 3.91±0.93, respectively (compared with CMV [100]), which were significantly higher than that of the empty vector group (0.199±0.20) (all P < 0.001). However, the rCela1(-205/+8) and hSPINK1 promoters exhibited no significant promoter activity. Compared to the CMV promoter, the ZsGreen expression levels driven by the hPRSS1 and rCela1(-500/+8) promoter were lower, whereas the pancreas-to-liver ZsGreen mRNA ratio demonstrated that both compacted promoters had pronounced pancreatic transduction preference, which elevated pancreatic-specific expression by 77.81- and 63.02-fold, respectively (all P < 0.001). We finally introduction of human amylase AMY2A (34 bp) and rat Amy2a3 (40 bp) enhancer into hPRSS1 and rCela1(-500/+8) promoters. The Amy2(rat)-hPRSS1 promoter markedly increased the pancreatic transduction efficiency by 8-fold, and was selected as the optimal compacted enhancer-promoter sequence.

Conclusion

The compact pancreatic acini-specific enhancer-promoter, Amy2(rat)-hPRSS1, dramatically improved the pancreas gene transfer efficiency in vivo and eliminating liver tropism, serving a promising tool for pancreas-specific gene therapy targeting exocrine pancreatic diseases, including acute pancreatitis, chronic pancreatitis, and precancerous lesions of pancreatic cancer.

References

1. Wang YC, Mao XT, Sun C, et al. Pancreas-directed AAV8-hSPINK1 gene therapy safely and effectively protects against pancreatitis in mice. Gut 2024;73:1142-1155.

Disclosure

All authors declare no conflicts of interest.

Introduction

Aryl hydrocarbon receptor (AHR) is a transcription factor that’s commonly upregulated in pancreatic ductal adenocarcinoma (PDAC). There are evidence that upregulated AHR increases cancer aggressiveness, invasiveness, migration and decreases survival rates. This can be linked to epithelial-mesenchymal transition, when cells acquire a migratory mesenchymal phenotype. AHR expression levels have been shown to influence epithelial or mesenchymal phenotype, however the exact mechanism is not yet know. EMT transcription factors (EMT-TF) (ZEB1, ZEB2, SNAI1, SNAI2, TWIST) can regulate EMT, but their relationship with AHR or the possibility to decrease PDAC migration and invasion by targeting AHR has not been widely studied.

Aims & Methods

The aim of this study was to investigate correlations of AHR and EMT-TF RNA expression in PDAC tissue, patient outcomes and the effects of AHR modulation on PDAC cell migration and invasion. Pancreatic carcinoma tissue and next-to-cancer tissue samples were obtained from 86 patients who underwent partial duodenopancreatectomy (Whipple resection) for pancreatic carcinoma. The presence of cancerous tissue in the samples was confirmed histologically. Cancerous and next-to-cancer tissues were taken from the same patients. Next-to-cancer tissues were used as reference controls. Total RNA was extracted after which it was converted to cDNA and RNA expression was analyzed using real time polymerase chain reaction using AHR, GAPDH, ZEB1, ZEB2, SNAI1, SNAI2 and TWIST primers. Expression changes were calculated using 2(-Delta Delta CT) method, correlations were calculated using Spearman method, Survival analysis was done using Log-Rank method. PDAC cells (BxPC-3; Su.86.86) were grown in RPMI medium with additives and treated for 24 hours with AHR inhibitor (BAY) after which they were seeded for cell migration analysis using scratch assay and 3D spheroid assays and invasiveness analysis using 3D spheroid method utilizing a basement membrane matrix.

Results

The results showed that AHR expression change in cancer compared to next-to-cancer tissue positively correlated with ZEB1 (r=0.801), SNAI1 (r=0.687) and SNAI2 (r=0.802) expression changes but did not correlate with ZEB2 or TWIST expression changes. Grouping the patient into AHR low (1/2 lowest AHR expression patients) and AHR high (1/2 highest AHR expression patients) showed similar results, with most EMT-TF relative expression being decreased compared to next-to-cancer tissue (AHR 0.19-fold, ZEB1 0.15-fold, ZEB2 0.62-fold, SNAI1 0.12-fold, SNAI2 0.21-fold, TWIST – no change). In AHR high group most of EMT-TF relative expression was increased (AHR 11.7-fold, ZEB1 6.6-fold, ZEB2 – no change, SNAI1 12.4-fold, SNAI2, 67.2-fold, TWIST – no change). Survival analysis of AHR low and AHR high groups revealed a significantly higher survival rates in AHR low group (54 months median) compared to AHR high group (19 months median). The results of PDAC cell line migration showed a statistically significant decrease (by 30-49 % depending on cell line and method) as well as decreased invasiveness (20-45 % depending on cell line).

Conclusion

AHR plays a role in PDAC patient survival possibly through its involvement with a number of EMT-TF. AHR inhibition could prove a viable strategy in reducing PDAC aggressiveness through EMT process modulation.