Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy associated with poor prognosis. Baicalein, a flavonoid extracted from the roots of Scutellaria baicalensis, traditionally used in Chinese medicine, has demonstrated potential in inhibiting cancer development and progression. However, its mechanism of action remains poorly understood, particularly regarding epigenetic gene regulation through m6A RNA methylation. In this study, three human PDAC cell lines and one nonmalignant cell line were employed. The effects of baicalein were examined using multiple assays, including RT-qPCR, MeRIP-qPCR, Western blotting, spheroid formation, RNA stability, and MTT, to evaluate cellular functions and m6A regulation. Baicalein significantly reduced cell viability, migration, invasion, and colony formation. It also downregulated FTO, an enzyme critical for m6A RNA demethylation. Knockdown of FTO replicated the effects of baicalein, underscoring its oncogenic role in PDAC. Bioinformatic analysis identified ZEB1-a key transcription factor in epithelial-to-mesenchymal transition-as an m6A-modified target regulated by FTO. Both baicalein treatment and FTO knockdown enhanced m6A modification and decreased ZEB1 mRNA stability, thereby suppressing stemness-related features. Rescue experiments further confirmed that baicalein disrupts the TGF-(3/FTO/ZEB1 signaling axis, highlighting its therapeutic potential in PDAC. This study offers fundamental insights for the development of novel therapeutic strategies targeting PDAC.

ABSTRACT Baicalein, a bioactive flavonoid derived from Scutellaria baicalensis, possesses notable anti‐inflammatory, antioxidative, and anticancer properties. Despite its therapeutic potential, the full scope of its effects on healthspan and longevity remains unexplored. This study investigates the impact of baicalein on longevity and health‐related biomarkers using the nematode Caenorhabditis elegans. Baicalein was administered to a wild‐type N2 strain, seven mutant strains, and three reporter strains. Its influence on longevity, motility, lipofuscin accumulation, and oxidative stress resistance was assessed. The methodology included Kaplan–Meier survival analysis, in vivo imaging, fluorescence microscopy, and real‐time PCR to evaluate RNA and protein expression. The findings indicate that baicalein significantly extends lifespan and enhances health markers, including improved motility, increased oxidative stress resistance, and reduced lipofuscin accumulation. Mechanistically, baicalein suppressed the DAF‐2‐mediated insulin/IGF‐1 signaling pathway and promoted the nuclear translocation of DAF‐16, a pivotal longevity transcription factor. Furthermore, baicalein upregulated the expression of the sod‐3 gene, which is associated with enhanced stress tolerance and lifespan extension. These results elucidate the function of baicalein in promoting longevity and healthspan in C. elegans through modulation of insulin/IGF‐1 signaling. Future studies are warranted to explore the applicability of baicalein in human aging to pave the way for innovative antiaging supplement formulations.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy associated with poor prognosis. Baicalein, a flavonoid extracted from the roots of Scutellaria baicalensis, traditionally used in Chinese medicine, has demonstrated potential in inhibiting cancer development and progression. However, its mechanism of action remains poorly understood, particularly regarding epigenetic gene regulation through m6A RNA methylation. In this study, three human PDAC cell lines and one nonmalignant cell line were employed. The effects of baicalein were examined using multiple assays, including RT-qPCR, MeRIP-qPCR, Western blotting, spheroid formation, RNA stability, and MTT, to evaluate cellular functions and m6A regulation. Baicalein significantly reduced cell viability, migration, invasion, and colony formation. It also downregulated FTO, an enzyme critical for m6A RNA demethylation. Knockdown of FTO replicated the effects of baicalein, underscoring its oncogenic role in PDAC. Bioinformatic analysis identified ZEB1-a key transcription factor in epithelial-to-mesenchymal transition-as an m6A-modified target regulated by FTO. Both baicalein treatment and FTO knockdown enhanced m6A modification and decreased ZEB1 mRNA stability, thereby suppressing stemness-related features. Rescue experiments further confirmed that baicalein disrupts the TGF-β/FTO/ZEB1 signaling axis, highlighting its therapeutic potential in PDAC. This study offers fundamental insights for the development of novel therapeutic strategies targeting PDAC.

Purpose. Surgical resection is the only curative option for pancreatic carcinoma, but disease-free and overall survival times after surgery are limited due to early tumor recurrence, most often originating from local microscopic tumor residues (R1 resection). The intraoperative identification of microscopic tumor residues within the resection margin in situ could improve surgical performance. The aim of this study was to evaluate the effectiveness of fiber-optic microscopy for detecting microscopic residues in vital pancreatic cancer tissues. Experimental Design. Fresh whole-mount human pancreatic tissues, histological tissue slides, cell culture, and chorioallantoic membrane xenografts were analyzed. Specimens were stained with selected fluorophore-conjugated antibodies and studied using conventional wide-field and self-designed multicolor fiber-optic fluorescence microscopy instruments. Results. Whole-mount vital human tissues and xenografts were stained and imaged using an in situ immunofluorescence protocol. Fiber-optic microscopy enabled the detection of epitope-based fluorescence in vital whole-mount tissue using fluorophore-conjugated antibodies and enabled visualization of microvascular, epithelial, and malignant tumor cells. Among the selected antigen-antibody pairs, antibody clones WM59, AY13, and 9C4 were the most promising for fiber-optic imaging in human tissue samples and for endothelial, tumor and epithelial cell detection. Conclusions. Fresh dissected whole-mount tissue can be stained using direct exposure to selected antibody clones. Several antibody clones were identified that provided excellent immunofluorescence imaging of labeled structures, such as endothelial, epithelial, or EGFR-expressing cells. The combination of in situ immunofluorescence staining and fiber-optic microscopy visualizes structures in vital tissues and could be proposed as an useful tool for the in situ identification of residual tumor mass in patients with a high operative risk for incomplete resection.

Introduction The maximum effect of the local ablation (radiofrequency and microwave therapy) of liver tumors is achieved when the ablation region completely encloses the tumor area. In the present study, the self-constructed system based on fiberoptic detection of tissue autofluorescence was used for identification of local extension of ablation area and of chemotherapy-induced local tumor necrosis.

Antibodies have gained considerable importance in laboratory and clinical settings. Currently, antibodies are extensively employed for the diagnosis and treatment of several human diseases. Herein, using targeted and cell immunisation approaches, we developed and characterised an antibody clone, DWH24. We found that DWH24 is an IgMκtype antibody that enables excellent visualisation and quantification of dead cells using immunofluorescence, fluorescence microscopy, and flow cytometry. This property was proved by the spontaneous cell death of several tumour cell lines and stimulated T cells, as well as after chemo- and photodynamic therapy. Unlike conventional apoptosis and cell death markers, DWH24 binding occurred in a Ca2+- and protein-independent manner and enabled live imaging of cell death progress, as shown using time-lapse microscopy. The binding specificity of DWH24 was analysed using a human proteome microarray, which revealed a complex response profile with very high spot intensities against various proteins, such as tropomyosin variants and FAM131C. Accordingly, DWH24 can be employed as a suitable tool for the cost-effective and universal analysis of cell death using fluorescence imaging and flow cytometry.

Silver has been in clinical use since ancient times and silver nanoparticles (AgNPs) have attracted attention in cancer therapy. We investigated the mechanisms by which AgNPs inhibit pancreatic ductal adenocarcinoma (PDAC). AgNPs were synthesized and 3 human PDAC and 2 nonmalignant primary cell lines were treated with AgNPs. MTT, MAPK, colony, spheroid and scratch assays, Western blotting, TEM, annexin V, 7-AAD, and H2DCFDA staining, FACS analysis, mRNA array and bioinformatics analyses, tumor xenograft transplantation, and immunohistochemistry of the treated cells were performed. We found that minimal AgNPs amounts selectively eradicated PDAC cells within a few hours. AgNPs inhibited cell migration and spheroid and colony formation, damaged mitochondria, and induced paraptosis-like cell death with the presence of cytoplasmic vacuoles, dilation of the ER and mitochondria, ROS formation, MAPK activity, and p62 and LC3b expression, whereas effects on the nucleus, DNA fragmentation, or caspases were not detectable. AgNPs strongly decreased tumor xenograft growth without side effects and reduced the expression of markers for proliferation and DNA repair, but upregulated paraptosis markers. The results highlight nanosilver as complementary agent to improve the therapeutic efficacy in pancreatic cancer.

Glucocorticoids (GCs) are widely used in tumor therapy to reduce tumor growth, inflammation, edema, and other side effects. Controversially, GCs may also cause the progression of highly aggressive pancreatic ductal adenocarcinoma (PDAC). Because microRNA (miR) and autophagy signaling support the invasive growth of PDAC, we asked whether these mechanisms may be targeted by GCs. Six established human PDAC cell lines, tissue from patients who received GC medication ( n = 35) prior to surgery, or not ( n = 35), and tumor xenografts were examined by RT‒qPCR, transmission electron microscopy (TEM), monodansylcadaverine (MDC) staining, immunohistochemistry, in situ hybridization, gene array and Kaplan‒Meier analysis with bioinformatics, and MTT, western blot, colony, spheroid, migration, and invasion assays. We found that various GCs, including dexamethasone (DEX), induced typical features of macroautophagy with the appearance of autolysosomes, enhanced LC3-II, decreased SQSTM1/p62 expression and induced epithelial-mesenchymal transition (EMT) and gemcitabine resistance. The GC receptor (GR) antagonist mifepristone (RU486) counteracted DEX-induced autophagy features, suggesting that the GC-GR complex is involved in the induction of autophagy. The autophagy-related miR-378i and miR-378a-3p were selected as the top upregulated candidates, and their high expression in PDAC patient tissue correlated with low survival. siRNA-mediated downregulation of miR-378 inhibited DEX-induced autophagy, and tumor progression. Bioinformatics confirmed the contribution of miR-378 to the regulation of signaling networks involved in GC-induced autophagy and tumor progression. The construction of a molecular docking model revealed stable binding of miR-378 to the DEX-GR complex, suggesting direct regulation. These substantial, novel, in-depth data reveal that GCs favor autophagy-mediated cancer progression by inducing miR-378 and GR binding and implicate GR and miR-378 as new therapeutic targets.

Several collagen subtypes are involved in pancreatic ductal adenocarcinoma (PDAC) desmoplasia, which constrains therapeutic efficacy. We evaluated collagen type VIII alpha 1 chain (COL8A1), whose function in PDAC is currently unknown. We identified COL8A1 expression in 7 examined PDAC cell lines by microarray analysis, western blotting, and RT‒qPCR. Higher COL8A1 expression occurred in 2 gemcitabine-resistant PDAC cell lines; pancreas tissue (n=15) from LSL-KrasG12D/+; p48-Cre mice with advanced PDAC predisposition; and PDAC parenchyma and stroma of a patient tissue microarray (n=82). Bioinformatic analysis confirmed higher COL8A1 expression in PDAC patient tissue available from TCGA (n=183), GTEx (n=167), and GEO (n=261) databases. siRNA or lentiviral sh-mediated COL8A1 inhibition in PDAC cells reduced migration, invasion and gemcitabine resistance and resulted in lower cytidine deaminase and thymidine kinase 2 expression and was rescued by COL8A1-secreting cancer-associated fibroblasts (CAFs). The activation of COL8A1 expression involved cJun/AP-1, as demonstrated by CHIP assay and siRNA inhibition. Downstream of COL8A1, activation of ITGB1 and DDR1 receptors and PI3K/AKT and NF-κB signaling occurred, as detected by expression, adhesion and EMSA binding studies. Orthotopic transplantation of PDAC cells with downregulated COL8A1 expression resulted in reduced tumor xenograft growth and lower gemcitabine resistance but was prevented by cotransplantation of COL8A1-secreting CAFs. Most importantly, COL8A1 expression in PDAC patient tissues from our clinic (n=84) correlated with clinicopathological data, and we confirmed these findings by the use of patient data (n=177) from the TCGA database. These findings highlight COL8A1 expression in tumor and stromal cells as a new biomarker for PDAC progression.

[This corrects the article DOI: 10.3389/fonc.2021.738801.].

Silver nanoparticles (AgNPs) have attracted attention in cancer therapy and might support the treatment of pancreatic ductal adenocarcinoma (PDAC). Silver is in clinical use in wound dressings, catheters, stents and implants. However, the side effects of systemic AgNP treatment due to silver accumulation limit its therapeutic application. We evaluated whether the antioxidant and natural agent α-lipoic acid might prevent these side effects. We synthesized AgNPs using an Ionic-Pulser® Pro silver generator and determined the concentration by inductively coupled plasma–optical emission spectrometry. The effect of α-lipoic acid was examined in four PDAC and two nonmalignant cell lines by MTT, FACS analysis, TEM, xenotransplantation and immunohistochemistry. The viability of PDAC cells was nearly totally abolished by AgNP treatment, whereas nonmalignant cells largely resisted. α-Lipoic acid prevented AgNP-induced cytotoxicity in nonmalignant cells but not in PDAC cells, which might be due to the higher sensitivity of malignant cells to silver-induced cytotoxicity. α-Lipoic acid protected mitochondria from AgNP-induced damage and led to precipitation of AgNPs. AgNPs reduced the growth of tumor xenografts, and cotreatment with α-lipoic acid protected chick embryos from AgNP-induced liver damage. Together, α-lipoic acid strongly reduced AgNP-induced side effects without weakening the therapeutic efficacy.

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor with a poor prognosis. More effective treatment options are urgently needed. The use of physical and weak alternating electric fields (TTFields) can inhibit cell division of PDAC carcinoma and is currently being investigated in clinical trials. Here, we analyzed this new physical treatment under non-ideal conditions such as may occur during patient treatment. Three established human PDAC cell lines BxPC-3, gemcitabine-resistant BxPC-3 (BxGem), AsPC-1, and a non-malignant primary pancreatic cell line CRL-4023 were treated with TTFields in vitro. MTT assays, electrical impedance measurement, cell staining with Annexin V/7AAD followed by FACS analysis, digital image analysis and immunohistochemistry were performed. Treatment with TTFields smaller than 0.7 V/cm and field lines in the direction of mitotic spindle orientation significantly inhibited proliferation of all PDAC cells at 150 kHz, but significantly increased viability of AsPC-1 cells at all frequencies between 100 kHz and 300 kHz and that of BxPC-3 and BxGem cells at 250 kHz. Apoptosis or necrosis were not induced. Non-malignant CRL-4023 cells were not affected at 150 kHz TTFields damaged PDAC cell lines but even favored their viability at very weak field strength and unfavorable frequency or inadequate field direction. (c) 2021 Elsevier B.V. All rights reserved.

Broccoli-derived isothiocyanate sulforaphane inhibits inflammation and cancer. Sulforaphane may support healthy aging, but the underlying detailed mechanisms are unclear. We used the C. elegans nematode model to address this question. Wild-type and 4 mutant C. elegans worm strains were fed in the presence or absence of sulforaphane and E. coli food bacteria transfected with RNA interference gene constructs. Kaplan–Meier survival analysis, live imaging of mobility and pharyngeal pumping, fluorescence microscopy, RT–qPCR, and Western blotting were performed. In the wild type, sulforaphane prolonged lifespan and increased mobility and food intake because of sulforaphane-induced upregulation of the sex-determination transcription factor TRA-1, which is the ortholog of the human GLI mediator of sonic hedgehog signaling. In turn, the tra-1 target gene daf-16 , which is the ortholog of human FOXO and the major mediator of insulin/IGF-1 and aging signaling, was induced. By contrast, sulforaphane did not prolong lifespan and healthspan when tra-1 or daf-16 was inhibited by RNA interference or when worms with a loss-of-function mutation of the tra-1 or daf-16 genes were used. Conversely, the average lifespan of C. elegans with hyperactive TRA-1 increased by 8.9%, but this longer survival was abolished by RNAi-mediated inhibition of daf-16 . Our data suggest the involvement of sulforaphane in regulating healthy aging and prolonging lifespan by inducing the expression and nuclear translocation of TRA-1/GLI and its downstream target DAF-16/FOXO.

Background: The exact identification of tumor boundaries and related liver segments is especially important for liver tumor surgery. This study aimed to evaluate a new approach for vascular boundary assessment and surgical navigation based on fiber-optic microscopy and microvascular fluorescence labeling. Methods: Antibody clones with fast binding ability were identified and selected using immunofluorescence. We evaluated the endothelial capture efficacy for an anti-mouse CD31 antibody labeled with different fluorophores and different degrees of labeling ex vivo. Segment boundary identification and navigation potential using endothelial capture were explored by two different fiber-optic microscopy systems. Finally, microvasculature labeling and fiber-optic microscopy were used to identify and treat microscopic liver tumors in vivo. Results: The following monoclonal antibodies were selected: anti-mouse CD31 (clone 390), anti-mouse CD54 (YN1/1.7.4), anti-human CD31 (WM59), and anti-human CD54 (HA58). These clones showed fast binding to endothelial cells and had long half-lives. The fluorophore choice and the degree of antibody labeling did not significantly affect capture efficacy in an isolated liver perfusion model. The microvascular system was clearly identified with wide-field fiber-optic microscopy after labeling the endothelium with low doses of specific antibodies, and the specifically labeled liver segment could be microscopically dissected. High antibody doses were required for confocal laser endomicroscopy. After microscopically identifying the vascular margin in vivo, tumor thermoablation strongly reduced tumor size or totally eliminated tumors. Conclusions: We demonstrated that vascular boundaries of liver tumors and locally perfused liver segments were accurately identified and surgical micronavigation was facilitated with fiber-optic microscopy and selected endothelium-specific antibodies.

OBJECTIVE:The aim of this research was to investigate if dielectric spectroscopy between 100 Hz and 1 MHz provides reliable information about water distribution in ischaemic heart tissue and to investigate the influence of temperature on oedema formation.METHODS:We examined hearts of landrace piglets (n = 13) during ischaemia at 35 °C, 25 °C, and 15 °C. The dielectric permittivity ε'(f) and conductivity σ'(f) were calculated from impedance spectra measured between 100Hz and 1MHz. Gap junction uncoupling (GJU) was identified in the sigmoidal time course of ε'(13 kHz). The extracellular space index (ECSI) was estimated by the ratio σ'(100 Hz)/σ'(1 MHz). Intercalated water was analysed in electron microscopy images of the myocardial samples and was used to calculate the extracellular space index ECSIhisto. The ECSI and ECSIhisto were compared during ischaemia. GJU and oedema formation were simulated with an electrical heart model.RESULTS:At the onset of ischaemia, the ECSI was significantly higher than the ECSIhisto (p < .01). GJU during ischaemia was temperature-dependent. After GJU, the values of the ECSI and ECSIhisto matched very well. The simulations confirmed the influence of GJU on the ECSI.SIGNIFICANCE:The estimation of cell oedema with the ECSI is reliable only after GJU. The development of oedema estimated by the ECSI was delayed at cooler temperatures.

Our aim was to investigate if the cardioplegic solution HTK can be improved by the addition of the ROS scavenger melatonin. 158 guinea pig hearts without (UI80) or with HTK protection (HTK80) were investigated in ischemia/reperfusion experiments. Ischemia lasted 80 min at 30 °C. Melatonin was given before ischemia (UI80 + M1, HTK80 + M1) or before and after ischemia (UI80 + M2, HTK80 + M2). We measured the left ventricular developed pressure (LVDP), diastolic pressure (LVPmin), cardiac rhythm (VC-RR), time of electrical cell uncoupling (t-in) and recovery (t-ret), intracellular Ca++ [Ca++], and postischemic ROS. After 45 min reperfusion, LVDP in UI80 was significantly higher than in HTK80 (p < .01). Compared to UI80, the postischemic ROS burst was slightly smaller in HTK80 and significantly smaller in HTK80 + M1 and HTK80 + M2 (p < .05). Melatonin had no effect on LVPmin, t-in, t-ret, [Ca++], and on LVDP in groups UI80 + M1 and HTK80 + M1, improved slightly VC-RR (n. s.) but significantly decreased LVDP in the groups UI80 + M2 and HTK80 + M2 (p < .01). With melatonin we were able to attenuate the postischemic ROS burst, but the tissue damage by ROS seemed to be less important for the chosen ischemia condition because melatonin was unable to improve the functional recovery during reperfusion of HTK protected hearts.

The therapy resistance of pancreatic cancer is associated with the loss of gap junction intercellular communication and connexin 43 expression. The broccoli isothiocyanate sulforaphane restored these features and therapy sensitivity. We investigated whether microRNA signaling is involved. Established cell lines and a patient tissue array (n = 96) were evaluated by miRNA and gene array, bioinformatics, expression studies, in situ hybridization and immunohistochemistry. Sulforaphane inhibited the expression of our top candidate miR30a-3p. Upon transfection of miR30a-3p inhibitors, the gemcitabine bystander effect, Cx43 expression and intercellular communication increased, whereas miR30a-3p mimics inhibited the luciferase activity of a Cx43 3'-UTR construct. miR30a-3p-overexpressing tumor xenografts had a decreased tumor volume and increased gemcitabine sensitivity. In patient tissues, a higher expression of miR30a-3p and a lower expression of Cx43 correlated with malignancy. These findings provide new knowledge and suggest sulforaphane as cotreatment for pancreatic cancer.

OBJECTIVE:We investigated canine hearts during ischemia after aortic cross clamping (UI, n  =  20) and after HTK-cardioplegia (HTK, n  =  24) at 35 °C, 25 °C, 15 °C, and 5 °C with the aim to compare tissue changes caused by the activity of anaerobic metabolism(AAM), cell membrane destruction(CD), and gap junction uncoupling(GJU). APPROACH:We measured continuously the complex dielectric spectrum(DS), ATP- and lactate content, extracellular pH, and rigor contracture. To identify changes in DS caused by AAM, CD, and GJU we performed additional experiments on the gap junction-free skeletal muscle. We used heart model simulations to calculate the effect of temperature. MAIN RESULTS:AAM affected the DS at 10 MHz and we found a strong correlation between DS and the proton concentration with a maximum of DS at 10 mmol g-1 dry weight in ATP-concentration. The time of GJU was detected by a characteristic increase in DS and CD by a characteristic decrease at 13 kHz. In comparison to UI, GJU, AAM and CD were delayed by HTK and by hypothermia, indicating a minimization of energy consumption and an improved preservation of tissue structure. SIGNIFICANCE:The novel findings were that in UI at 5 °C GJU occurred earlier and AAM remained constant, indicating a less effective preservation in UI by deep hypothermia in contrast to HTK.