SMAD4 endows TGF-ß1-induced highly invasive tumor cells with ferroptosis vulnerability in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive malignancy prone to recurrence and metastasis. Studies show that tumor cells with increased invasive and metastatic potential are more likely to undergo ferroptosis. SMAD4 is a critical molecule in the transforming growth factor ß (TGF-ß) pathway, which affects the TGF-ß-induced epithelial-mesenchymal transition (EMT) status. SMAD4 loss is observed in more than half of patients with PDAC. In this study, we investigated whether SMAD4-positive PDAC cells were prone to ferroptosis because of their high invasiveness. We showed that SMAD4 status almost determined the orientation of transforming growth factor ß1 (TGF-ß1)-induced EMT via the SMAD4-dependent canonical pathway in PDAC, which altered ferroptosis vulnerability. We identified glutathione peroxidase 4 (GPX4), which inhibited ferroptosis, as a SMAD4 down-regulated gene by RNA sequencing. We found that SMAD4 bound to the promoter of GPX4 and decreased GPX4 transcription in PDAC. Furthermore, TGF-ß1-induced high invasiveness enhanced sensitivity of SMAD4-positive organoids and pancreas xenograft models to the ferroptosis inducer RAS-selective lethal 3 (RSL3). Moreover, SMAD4 enhanced the cytotoxic effect of gemcitabine combined with RSL3 in highly invasive PDAC cells. This study provides new ideas for the treatment of PDAC, especially SMAD4-positive PDAC.

Maximum Value on Arterial Phase Computed Tomography Predicts Prognosis and Treatment Efficacy of Sunitinib for Pancreatic Neuroendocrine Tumours.

PURPOSE: This study was designed to assess the computed tomography maximum (CTmax) value on pretherapeutic arterial phase computed tomography (APCT) images to predict pancreatic neuroendocrine tumours (pNETs) recurrence and clarify its role in predicting the outcome of tumour therapy. METHODS: This retrospective study enrolled 250 surgical patients and 24 nonsurgical patients with sunitinib-based treatment in our hospital from 2008 to 2019. CT images were assessed, the maximum value was defined as "CTmax," and recurrence-free survival (RFS) or progression-free survival (PFS) was compared between a high-CTmax group and a low-CTmax group among patients who underwent surgical resection or nonsurgical, sunitinib-based treatment according to the CTmax cutoff value. RESULTS: In ROC curve analysis, a CTmax of 108 Hounsfield units, as the cutoff value, achieved an AUC of 0.796 in predicting recurrence. Compared with the low-CTmax group, the high-CTmax group had a longer RFS (p < 0.001). Low CTmax was identified as an independent factor for RFS (p < 0.001) in multivariate analysis; these results were confirmed using the internal validation set. The CTmax value was significantly correlated with the microvascular density (MVD) value (p < 0.001) and the vascular endothelial growth factor receptor 2 (VEGFR2) score (p < 0.001). Furthermore, the high-CTmax group had a better PFS than the low-CTmax group among the sunitinib treatment group (p = 0.007). CONCLUSIONS: The tumour CTmax on APCT might be a potential and independent indicator for predicting recurrence in patients who have undergone surgical resection and assessing the efficacy of sunitinib for patients with advanced metastatic pNETs.

Hernandezine induces autophagic cell death in human pancreatic cancer cells via activation of the ROS/AMPK signaling pathway.

Hernandezine (Her) is a bisbenzylisoquinoline alkaloid extracted from the traditional Chinese herbal medicine Thalictrum glandulosissimum. Evidence shows that Her is a natural agonist of adenosine monophosphate (AMP)-activated protein kinase (AMPK) and induces apoptosis and autophagy in tumor cells. In this study, we investigated the role of autophagy in Her-induced cell death in human pancreatic cancer cell lines. We showed that Her dose-dependently suppressed cell proliferation, promoted autophagy and induced autophagic death in pancreatic ductal adenocarcinoma (PDAC) cell lines Capan-1 and SW1990. The IC50 values of Her in inhibition of Capan-1 and SW1990 cells were 47.7 µM and 40.1 µM, respectively. Immunoblotting showed that Her (1-40 µM) promoted the conversion of LC3-I to LC3-II, and Her exerted concentration-dependent and time-dependent effects on autophagy activation in PDAC cells. In transmission electron microscopy and fluorescence image analysis, we found that autophagic vacuoles were significantly increased in Her-treated cells. Knockdown of ATG5, a key gene in the autophagy pathway, alleviated the activation of autophagy by Her. These results demonstrated that Her induced autophagy in PDAC cells. Intensely activated autophagy could promote cell death. The autophagy inhibitors, BafA1 and HCQ significantly inhibited Her-induced cell death, implying that Her induced autophagic cell death in PDAC cells. Moreover, we showed that Her activated autophagy by increasing the phosphorylation of AMPK and decreasing the phosphorylation of mTOR/p70S6K. Knockdown of AMPKα relieves the autophagic cell death induced by Her. Furthermore, Her concentration-dependently enhanced reactive oxygen species (ROS) generation in PDAC cells. Antioxidants could reduce the phosphorylation of AMPK and suppress autophagic cell death induced by Her. Our study provides evidence for the development of Her as a therapeutic agent for the treatment of pancreatic cancer.

Probiotic and Safety Evaluation of Twelve Lactic Acid Bacteria as Future Probiotics.

The human gut flora is highly diverse. Most lactic acid bacteria (LAB) are widely used as probiotics in human and animal husbandry and have a variety of physiological benefits. This article mainly studied the bacteriostatic ability of LAB against four pathogenic bacteria, gastrointestinal environment tolerance, and adhesion ability to Caco-2 cells. The genome of Lactiplantibacillus plantarum L461 was sequenced and analyzed. The results showed that strains F512, L461, and D469 had the most significant inhibitory effects on Escherichia coli, Salmonella enterica B, Staphylococcus aureus, and Listeria monocytogenes. In addition, strains L461, C502, and P231 showed good tolerance after exposure to simulated gastric fluid for 0-4 h. Strains C502, H781, and L461 showed good tolerance in simulated intestinal fluid. Strains L461 and H781 showed good adhesion to Caco-2 cells. The number of viable bacteria was more than 60. Therefore, we screened L. plantarum L461 from 12 LAB strains through three aspects of evaluation, and conducted whole genome sequencing and analysis. Sequencing results showed that L. plantarum L461 had more defense mechanisms and phage annotation genes than L. plantarum WCFS1. Virulence factor studies showed that L. plantarum L461 has iron absorption system and adhesion-related gene annotation, indicating that L. plantarum L461 has survival advantage in intestinal tract. The predicted results showed that there were eight phages with phage resistance in L. plantarum L461. L. plantarum L461 is sensitive to several antibiotics, notably penicillin and oxacillin. In summary, the results of this study prove that L. plantarum L461 has good prebiotic function and is safe. Therefore, L. plantarum L461 can be safely used as a potential functional probiotic.

MEN1 promotes ferroptosis by inhibiting mTOR-SCD1 axis in pancreatic neuroendocrine tumors.

Pancreatic neuroendocrine tumor (pNET) is the second most common malignant tumors of the pancreas. Multiple endocrine neoplasia 1 ( MEN1) is the most frequently mutated gene in pNETs and MEN1-encoded protein, menin, is a scaffold protein that interacts with transcription factors and chromatin-modifying proteins to regulate various signaling pathways. However, the role of MEN1 in lipid metabolism has not been studied in pNETs. In this study, we perform targeted metabolomics analysis and find that MEN1 promotes the generation and oxidation of polyunsaturated fat acids (PUFAs). Meanwhile lipid peroxidation is a hallmark of ferroptosis, and we confirm that MEN1 promotes ferroptosis by inhibiting the activation of mTOR signaling which is the central hub of metabolism. We show that stearoyl-coA desaturase (SCD1) is the downstream of MEN1-mTOR signaling and oleic acid (OA), a metabolite of SCD1, recues the lipid peroxidation caused by MEN1 overexpression. The negative correlation between MEN1 and SCD1 is further verified in clinical specimens. Furthermore, we find that BON-1 and QGP-1 cells with MEN1 overexpression are more sensitive to everolimus, a widely used drug in pNETs that targets mTOR signaling. In addition, combined use everolimus with ferroptosis inducer, RSL3, possesses a more powerful ability to kill cells, which may provide a new strategy for the comprehensive therapy of pNETs.

ALDOA inhibits cell cycle arrest induced by DNA damage via the ATM-PLK1 pathway in pancreatic cancer cells.

BACKGROUND: ALDOA is a glycolytic enzyme found mainly in developing embryos, adult muscle and various malignant tumours, including pancreatic tumours. Our previous study revealed that ALDOA, an oncogene, can promote the proliferation and metastasis of pancreatic tumours. Furthermore, ALDOA could predict poor prognosis in patients with pancreatic tumours. METHODS: IHC analysis of PDAC tissues was conducted. Western blotting, PCR, cellular IF experiments and cell cycle assessment were conducted utilizing cell lines. GSEA and KEGG pathway analysis were used to identify potential downstream pathways. RESULTS: To explore the effects of ALDOA on the occurrence and development of pancreatic tumours, we analysed the RNA sequencing results and found that ALDOA could inhibit the DDR. Under normal circumstances, when DNA is damaged, initiation of the DDR causes cell cycle arrest, DNA repair or cell apoptosis. Further experiments showed that ALDOA could inhibit DNA repair and reverse cell cycle arrest induced by DNA damage so that DNA damage persisted to promote the occurrence and progression of cancer. CONCLUSIONS: Regarding the molecular mechanism, we found that ALDOA inhibited the DDR and improved activation of the cell cycle checkpoint PLK1 by suppressing ATM, which promotes tumour cell progression. Consequently, ALDOA has a profound effect on pancreatic cancer development.

Synthesis and biological evaluation of 3-arylcoumarin derivatives as potential anti-diabetic agents.

A variety of substituted 3-arylcoumarin derivatives were synthesised through microwave radiation heating. The method has characteristics of environmental friendliness, economy, simple separation, and purification process, less by-products and high reaction yield. Those 3-arylcoumarin derivatives were screened for antioxidant, α-glucosidase inhibitory and advanced glycation end-products (AGEs) formation inhibitory. Most compounds exhibited significant antioxidant and AGEs formation inhibitory activities. Anti-diabetic activity studies showed that compounds 11 and 17 were equipotent to the standard drug glibenclamide in vivo. According to the experimental results, the target compound 35 can be used as a lead compound for the development of new anti-diabetic drugs. The whole experiment showed that anti-diabetic activity is prevalent in 3-arylcoumarins, which added a new natural skeleton to the development of anti-diabetic active drugs.

Synthesis and biological evaluation of 3-arylcoumarins as potential anti-Alzheimer's disease agents.

Alzheimer's disease, a neurodegenerative illness, has the extremely complex pathogenesis. Accumulating evidence indicates there is a close relationship between several enzymes and Alzheimer's disease. Various substituted 3-arylcoumarin derivatives were synthesised, and their in vitro activity, including cholinesterase inhibitory activity, monoamine oxidase inhibitory activity, and antioxidant activity were investigated. Most of the compounds exhibited high activity; therefore 3-arylcoumarin compounds have the potential as drug candidates for the treatment of Alzheimer's disease.

Synthesis and biological evaluation of 3-(4-aminophenyl)-coumarin derivatives as potential anti-Alzheimer's disease agents.

The work is focused on the design of drugs that prevent and treat Alzheimer's disease (AD) and its complications. A series of 3-(4-aminophenyl)-coumarin derivatives designed, synthesised, fully characterised and evaluated in vitro/vivo. The biological assay experiments showed that some compounds displayed a clearly selective inhibition for acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Among all compounds, compound 4m exhibited the highest AChE inhibition with an IC50 value of 0.091 ± 0.011 µM and compound 4k exhibited the highest BuChE inhibition with an IC50 value of 0.559 ± 0.017 µM. A zebrafish behaviour analyser (Zebrobox) was used to determine the behavioural effects of the active compound on the movement distance of the aluminium chloride-induced zebrafish. Compound 4m offered a potential drug design concept for the development of therapeutic or preventive agents for AD and its complications.

Transcription factor EB reprograms branched-chain amino acid metabolism and promotes pancreatic cancer progression via transcriptional regulation of BCAT1.

Pancreatic cancer cells have a much higher metabolic demand than that of normal cells. However, the abundant interstitium and lack of blood supply determine the lack of nutrients in the tumour microenvironment. Although pancreatic cancer has been reported to supply extra metabolic demand for proliferation through autophagy and other means, the specific regulatory mechanisms have not yet been elucidated. In this study, we focused on transcription factor EB (TFEB), a key factor in the regulation of autophagy, to explore its effect on the phenotype and role in the unique amino acid utilisation pattern of pancreatic cancer cells (PCCs). The results showed that TFEB, which is generally highly expressed in pancreatic cancer, promoted the proliferation and metastasis of PCCs. TFEB knockdown inhibited the proliferation and metastasis of PCCs by blocking the catabolism of branched-chain amino acids (BCAAs). Concerning the mechanism, we found that TFEB regulates the catabolism of BCAAs by regulating BCAT1, a key enzyme in BCAA metabolism. BCAA deprivation alone did not effectively inhibit PCC proliferation. However, BCAA deprivation combined with eltrombopag, a drug targeting TFEB, can play a two-pronged role in exogenous supply deprivation and endogenous utilisation blockade to inhibit the proliferation of pancreatic cancer to the greatest extent, providing a new therapeutic direction, such as targeted metabolic reprogramming of pancreatic cancer.

The stromal microenvironment endows pancreatic neuroendocrine tumors with spatially specific invasive and metastatic phenotypes.

Cancer-associated fibroblasts (CAFs) play an important role in a variety of cancers. However, the role of tumor stroma in nonfunctional pancreatic neuroendocrine tumors (NF-PanNETs) is often neglected. Profiling the heterogeneity of CAFs can reveal the causes of malignant phenotypes in NF-PanNETs. Here, we found that patients with high stromal proportion had poor prognosis, especially for that with infiltrating stroma (stroma and tumor cells that presented an infiltrative growth pattern and no regular boundary). In addition, myofibroblastic CAFs (myCAFs), characterized by FAP+ and α-SMAhigh, were spatially closer to tumor cells and promoted the EMT and tumor growth. Intriguingly, only tumor cells which were spatially closer to myCAFs underwent EMT. We further elucidated that myCAFs stimulate TGF-ß expression in nearby tumor cells. Then, TGF-ß promoted the EMT in adjacent tumor cells and promoted the expression of myCAFs marker genes in tumor cells, resulting in distant metastasis. Our results indicate that myCAFs cause spatial heterogeneity of EMT, which accounts for liver metastasis of NF-PanNETs. The findings of this study might provide possible targets for the prevention of liver metastasis.

Long-term intermittent hypoxia induces anxiety-like behavior and affects expression of orexin and its receptors differently in the mouse brain.

Studies have revealed a possible connection between orexin, narcolepsy, and obstructive sleep apnea (OSA). Orexin has an important role in the maintenance of arousal and wakefulness/sleeping states. To better understand the pathophysiological mechanism of OSA, we used a chronic intermittent hypoxia (CIH) model in mice to mimic OSA. In this way, we explored the effect of CIH on the locomotor activity and orexin system in the hypothalamus, cerebral cortex, and brainstem of mice. Male C57BL/6 J mice (8 weeks) in the CIH group were exposed in a hypoxia chamber for 8 h/day for 28 weeks. The re-oxygenation groups comprised the W2 group and W4 group, which were exposed to 28 weeks of CIH followed by 2 weeks and 4 weeks of re-oxygenation, respectively. The open field test was undertaken to observe locomotor activity. mRNA expression of orexin, orexin receptor type 1 (OX1R), and OX2R mRNA was evaluated by real-time reverse transcription-quantitative polymerase chain reaction. Mice subjected to long-term CIH exhibited significant anxiety-like behavior during the light period, and this behavior lasted until 4 weeks of re-oxygenation. mRNA expression of orexin was upregulated in the hypothalamus. mRNA expression of OX1R mRNA in the cerebral cortex and brainstem was downregulated by CIH. Two weeks and 4 weeks of re-oxygenation could not reverse these alternations. Long-term CIH may induce anxiety-like behavior and re-oxygenation cannot reverse these behavior. Moreover, OX1R has a significant role in the anxiety-related symptoms observed in long-term CIH.

Nuclear receptor coactivator 6 (NCoA6) promotes cell proliferation, migration, and invasion in pancreatic cancer.

BACKGROUND: Nuclear receptor coactivator 6 (NCoA6) is overexpressed in various cancers and considered a multifunctional coactivator of various transcription factors and nuclear receptors. However, the role of NCoA6 in pancreatic ductal adenocarcinoma (PDAC) remains unclear. METHODS: NCoA6 expression data in PDAC were extracted from TCGA and GTEx databases, and their correlation with survival outcomes were analyzed using the Kaplan-Meier plotter database. NCoA6 protein expression in PDAC tissues was evaluated using immunohistochemistry. RNA-sequencing technology was used to sequence the transcriptome of NCoA6-silenced PANC-1 cells, followed by differential expression, GO/KEGG and GSEA analyses. The effects of NCoA6 on cell proliferation, migration, invasion, cell cycle, and apoptosis were determined in two representative cell lines (PANC-1 and SW1990). Western blotting, qPCR, and co-immunoprecipitation were performed to explore the mechanism of action of NCoA6 in PDAC cells. RESULTS: NCoA6 expression was markedly increased in PDAC tissues, and high NCoA6 expression was associated with poor survival prognosis. However, there was no significant relationship between NCoA6 expression and metastasis in PDAC patients. Our RNA-sequencing data analysis found 1194 significant differentially expressed genes between the control and NCoA6-silenced PANC-1 cells. GO/KEGG analysis results mainly focused on cytokine production, cytokine activity, and cytokine-cytokine receptor interactions. GSEA results showed that the knockdown of NCoA6 affected the expression of histone deacetylase 1 (HDAC1) targeted genes. NCoA6 knockdown suppressed proliferation, migration, and invasion of PDAC cells. Finally, western blotting, qPCR, and co-immunoprecipitation results showed that NCoA6 interacted with HDAC1 and that NCoA6 expression was negatively correlated with F-box and WD repeat domain-containing 7 (FBW7) and caudal-related homeobox transcription factor 2 (CDX2) expression in pancreatic cancer. CONCLUSIONS: NCoA6 has a profound effect on cell proliferation, migration, invasion, and prognosis of PDAC and is potentially related to the expression of HDAC1, FBW7, and CDX2. Our results may provide novel therapeutic strategies for PDAC patients.

The Surgical and Therapeutic Activities of Non-Functional Pancreatic Neuroendocrine Tumors at a High-Volume Institution.

BACKGROUND: This study aimed to summarize the surgical and therapeutic activities of non-functional pancreatic neuroendocrine tumors (NF-PanNETs) and perform survival analyses of a 15-year single-institutional cohort of NF-PanNETs. METHODS: In total, 1001 patients with neuroendocrine neoplasms treated at Fudan University Shanghai Cancer Center were screened for inclusion, and 509 patients with NF-PanNETs from 2006 to 2020 were included. For time trend analyses, the 15-year study period was randomly divided into three periods. Survival analyses used the Kaplan-Meier method and Cox regression models. RESULTS: The total number of resected NF-PanNETs increased over the 15-year study period, from 5 resections in 2006 to 94 resections in 2020. A significant decrease in the tumor size was observed, from a mean of 4.0 cm to 3.3 cm, and to 3.0 cm in the most recent period (p = 0.006). Minimally invasive techniques gradually increased from 3.5% to 12.9%, and finally to 46.4% in the most recent period (p < 0.001). In non-metastatic and resected tumors, the tumor size (p < 0.001), positive lymph node (p < 0.001), adjuvant treatment (p = 0.048), and tumor grade (p < 0.001) were independent prognostic factors for recurrence-free survival (RFS). The microvascular invasion (p = 0.024) and tumor grade (p = 0.013) were independent prognostic factors for overall survival (OS). A malignant transformation from NET into neuroendocrine carcinoma was observed. CONCLUSIONS: An increasing number of NF-PanNETs resection and minimally invasive surgery was shown. In non-metastatic and resected tumors NF-PanNETs, tumor size, positive lymph node, adjuvant treatment, and tumor grade were independent predictors of RFS. Microvascular invasion and tumor grade were independent prognostic factors for OS.

Role of Somatostatin Receptor 2 in Nonfunctional Pancreatic Neuroendocrine Tumors: Clinicopathological Analysis of 223 Cases and Whole Exome Sequencing of a Multifocal Case.

OBJECTIVES: Somatostatin receptors are commonly expressed in most pancreatic neuroendocrine tumors (pNETs), a rare type of pancreatic tumors with high heterogeneity. However, the role of somatostatin receptor 2 (SSTR2) has seldom been investigated separately in pNET. This retrospective study aims to evaluate the role of SSTR2 in the clinicopathological features and genomic background of nonfunctional and well-differentiated pNET. METHODS: A total of 223 cases of nonfunctional well-differentiated pNET were included, and the correlation between SSTR2 status and clinicopathological outcome was evaluated. In addition, we performed whole exome sequencing in SSTR2-positive and SSTR2-negative pNETs and identified that the 2 lesions harbored different mutational landscapes. RESULTS: Negative SSTR2 immunochemistry staining was significantly related to an earlier onset of disease, larger tumor size, advanced stage of American Joint Committee on Cancer, and tumor metastasis in lymph nodes and liver. Under pathological assessment, positive peripheral aggression, vascular invasion, and perineural invasion were markedly increased in SSTR2-negative cases. Moreover, SSTR2-negative patients exhibited significantly worse progression-free survival than SSTR2-positive patients (hazard ratio, 0.23; 95% confidence interval, 0.10-0.53; P = 0.001). CONCLUSIONS: Somatostatin receptor 2-negative nonfunctional pNET might represent a subtype of pNET with poor outcomes and evolve from a different genomic background.

Role of Somatostatin Receptor in Pancreatic Neuroendocrine Tumor Development, Diagnosis, and Therapy.

Pancreatic neuroendocrine tumors (pNETs) are rare and part of the diverse family of neuroendocrine neoplasms (NENs). Somatostatin receptors (SSTRs), which are widely expressed in NENs, are G-protein coupled receptors that can be activated by somatostatins or its synthetic analogs. Therefore, SSTRs have been widely researched as a diagnostic marker and therapeutic target in pNETs. A large number of studies have demonstrated the clinical significance of SSTRs in pNETs. In this review, relevant literature has been appraised to summarize the most recent empirical evidence addressing the clinical significance of SSTRs in pNETs. Overall, these studies have shown that SSTRs have great value in the diagnosis, treatment, and prognostic prediction of pNETs; however, further research is still necessary.

Natural source, bioactivity and synthesis of benzofuran derivatives.

Benzofuran compounds are a class of compounds that are ubiquitous in nature. Numerous studies have shown that most benzofuran compounds have strong biological activities such as anti-tumor, antibacterial, anti-oxidative, and anti-viral activities. Owing to these biological activities and potential applications in many aspects, benzofuran compounds have attracted more and more attention of chemical and pharmaceutical researchers worldwide, making these substances potential natural drug lead compounds. For example, the recently discovered novel macrocyclic benzofuran compound has anti-hepatitis C virus activity and is expected to be an effective therapeutic drug for hepatitis C disease; novel scaffold compounds of benzothiophene and benzofuran have been developed and utilized as anticancer agents. Novel methods for constructing benzofuran rings have been discovered in recent years. A complex benzofuran derivative is constructed by a unique free radical cyclization cascade, which is an excellent method for the synthesis of a series of difficult-to-prepare polycyclic benzofuran compounds. Another benzofuran ring constructed by proton quantum tunneling has not only fewer side reactions, but also high yield, which is conducive to the construction of complex benzofuran ring systems. This review summarizes the recent studies on the various aspects of benzofuran derivatives including their important natural product sources, biological activities and drug prospects, and chemical synthesis, as well as the relationship between the bioactivities and structures.