Proliferation Of PANC-1 Cells Research Articles

Islet amyloid polypeptide does not suppress pancreatic cancer

ObjectivesPancreatic cancer risk is elevated approximately two-fold in type 1 and type 2 diabetes. Islet amyloid polypeptide (IAPP) is an abundant beta-cell peptide hormone that declines with diabetes progression. IAPP has been reported to act as a tumour-suppressor in p53-deficient cancers capable of regressing tumour volumes. Given the decline of IAPP during diabetes development, we investigated the actions of IAPP in pancreatic ductal adenocarcinoma (PDAC; the most common form of pancreatic cancer) to determine if IAPP loss in diabetes may increase the risk of pancreatic cancer. MethodsPANC-1, MIA PaCa-2, and H1299 cells were treated with rodent IAPP, and the IAPP analogs pramlintide and davalintide, and assayed for changes in proliferation, death, and glycolysis. An IAPP-deficient mouse model of PDAC (Iapp−/−; Kras+/LSL-G12D; Trp53flox/flox; Ptf1a+/CreER) was generated for survival analysis. ResultsIAPP did not impact glycolysis in MIA PaCa-2 cells, and did not impact cell death, proliferation, or glycolysis in PANC-1 cells or in H1299 cells, which were previously reported as IAPP-sensitive. Iapp deletion in Kras+/LSL-G12D; Trp53flox/flox; Ptf1a+/CreER mice had no effect on survival time to lethal tumour burden. ConclusionsIn contrast to previous reports, we find that IAPP does not function as a tumour suppressor. This suggests that loss of IAPP signalling likely does not increase the risk of pancreatic cancer in individuals with diabetes.

LHPP Inhibits the Viability, Migration, and Proliferation of PDAC Cells and Significantly Affects the Expression of SDC1 and S100p.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with a poor response to chemotherapy and an extremely poor prognosis. Recent studies have revealed that phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) can inhibit the growth of various cancers. Therefore, the current study was conducted to investigate the antitumor effects of LHPP in PDAC and to explore its mechanism using proteomics analysis. Immunohistochemical analysis of clinical samples demonstrated that LHPP expression levels were lower in tumor tissues compared to adjacent nontumor tissues. Moreover, multivariate COX regression analysis showed that LHPP expression level was an independent prognostic factor for the patients with PDAC. Patients with high LHPP expression had a better prognosis. The lentiviral vectors for normal control (NC), LHPP knockdown (KD), and LHPP overexpression (OE) were infected with BxPC-3 and PANC-1 cell lines. Cell counting kit-8 assay, Transwell assay, and flow cytometry analyses showed that LHPP overexpression significantly inhibited the cell viability, migration, and proliferation of BxPC-3 and PANC-1 cells. Moreover, xenograft tumor model demonstrated that LHPP overexpression inhibited xenograft tumor growth in vivo. Subsequently, proteins with significantly altered expression in BxPC-3 cells after lentivirus infection were detected using proteomics analyses. Interestingly, compared to the NC group, the expression of Syndecan 1 (SDC1) was significantly upregulated in the KD group, while that of S100P was significantly downregulated in the OE group. LHPP might emerge as an important target for delaying the advancement of PDAC, thereby providing a novel therapeutic approach for the treatment of PDAC.

BNIP3 enhances pancreatic cancer cell migration and proliferation via modulating autophagy under hypoxia

Chemotherapy and immunotherapy for pancreatic ductal adenocarcinoma (PDAC) have limited success for the intricated surrounding cancer microenvironment. Hypoxic microenvironment in PDAC causes the activation of multiple different molecules and signaling pathways compared with normoxia. We studied the roles of BNIP3 for the migration and proliferation of PDAC and Panc1 cells in vitro. In the present study, we found that BNIP3 expression was elevated and enhanced the migration and proliferation of CFPAC-1 and Panc1 cells under hypoxia. The upregulation of BNIP3 was important for the autophagic activation, while inhibition of autophagy with siRNA targeting Atg5 and Atg7 impaired the hypoxia-induced cell migration and proliferation. Additionally, blocking ERK1/2 mitogen-activated protein kinase (MAPK) signaling with PD98058 significantly down-regulated BNIP3 expression, autophagic activation, as well as the migration and proliferation of CFPAC-1 and Panc1 cells under hypoxia. Collectively, our results here uncover a hitherto unknown hypoxia-BNIP3-autophagy axis in modulating the migration and proliferation and provide a potential intriguing drug target for the therapy of PDAC.

MiR-203 suppresses pancreatic cancer cell proliferation and migration by modulating DUSP5 expression

BackgroundPancreatic cancer (PC) is an insidious cancer that is commonly diagnosed in advanced stages. Therefore, it is necessary to understand PC-related mechanisms in order to discover new and reliable diagnostic biomarkers. It is known that miRNAs play a crucial role in carcinogenesis by targeting mRNAs. In this study we aimed to explore interaction between downregulated miR-203 and its upregulated target DUSP5 in PC. MethodsUsing bioinformatics approaches we identified the DUSP5 as a direct target gene of miR-203 and detected potential binding sites between miR-203 and DUSP5. Additionally, we evaluated subcellular location, expression level and prognostic value of DUSP5 in PC through using various bioinformatics tools. To investigate the relationship between miR-203 and DUSP5, we increased the expression levels of miR-203 by transfecting miR-203 mimics into the pancreatic cancer cell line, PANC-1. Finally, MTT, wound healing, and colony formation assays were performed to determine effect of overexpressed miR-203 on proliferation and migration of PANC-1 cells. ResultsWe found that expression level of DUSP5 in pancreas tissue was one of the lowest tissue expression among all normal human tissue types. In addition, DUSP5 expression was upregulated both PC tissues and cell line and associated with poor overall survival in PC. Overexpression of miR-203 significantly downregulated expression level of DUSP5 and remarkably suppressed proliferation, migration and colony formation ability of PANC-1 cells. ConclusionsThese findings suggest that miR-203 restrains proliferation and migration of PC cells by regulating oncogenic activity of DUSP5 in PC, thereby could be novel candidate biomarkers for PC diagnosis and treatment.

MiR-24-3p Regulates Epithelial–Mesenchymal Transition and the Malignant Phenotype of Pancreatic Adenocarcinoma by Regulating ASF1B Expression

Anti-silencing function protein 1 homolog B (ASF1B) has been implicated in the occurrence and development of cancers. The present work explored the functional role and the expression regulation of ASF1B in pancreatic ductal adenocarcinoma (PDAC). Based on the real-time quantitative PCR (qRT-PCR) and immunohistochemistry (IHC), ASF1B was significantly upregulated in PDAC tissues. High expression of ASF1B was associated with a poor overall survival (OS) and recurrence-free survival (DFS) in the PDAC patients. ASF1B also showed a relatively higher expression in PDAC cells (AsPC-1, PANC-1) when compared with human pancreatic ductal epithelial cells (HPDFe-6). CCK8 and clone formation assay demonstrated that silencing ASF1B impaired the proliferation in PANC-1 and AsPC-1 cells, and Annexin V-PI staining showed an increased level of apoptosis upon ASF1B silencing. ASF1B silencing also suppressed the migration and invasion in PDAC cells, as revealed by Transwell assays. We further showed that miR-24-3p was downregulated in PDAC tissues and cells, which functionally interacted with ASF1B by dual-luciferase reporter assay. miR-24-3p negatively regulated ASF1B expression to modulate the malignant phenotype of PDAC cells. ASF1B shows high expression in PDAC, which promotes the malignancy and EMT process of PDAC cells. miR-24-3p is a negative regulator of ASF1B and is downregulated in PDAC cells. Our data suggest that targeting ASF1B/miR-24-3p axis may serve as an intervention strategy for the management of PDAC.

CircPUM1 Activates the PI3K/AKT Signaling Pathway by Sponging to Promote the Proliferation, Invasion and Glycolysis of Pancreatic Cancer.

Our study seeks to obtain data to assess the impact of circPUM1 on pancreatic cancer (PC) and its mechanism. The expression of circPUM1 and miR-200c-3p in PC and normal tissues and PC cell lines was collected and detected, and subsequently dual-luciferase assay-based verification of the binding site of the two was carried out. After interfering with circPUM1 expression in MIAPaCa-2 and PANC-1 cells, cell proliferation, viability, apoptosis rate, invasion ability, glucose consumption, and lactate production were measured by MTT, colony formation, flow cytometry, Transwell assays, and glucose and lactate assay kits. Additionally, western blot was utilized for assessing PI3K/AKT signaling pathway-related proteins. From the results, highly expressed circPUM1 and miR-200c-3p in PC tissues and cells were proved. Down-regulation of circPUM1 expression significantly inhibited cell proliferation, cell viability, invasion and glycolysis, while increasing the apoptosis rate. Down-regulated circPUM1 led to the inhibition of the PI3K/AKT signaling pathway activity in PC cells; while up-regulated circPUM1 increased its activity. Further experiments revealed that down-regulation of miR-200c-3p expression reversed the inhibitory effect of lowly expressed circPUM1 on PC cells. In summary, circPUM1 activates PI3K/AKT signaling pathway by sponging miR-200c-3p and promotes PC progression.

Proscillaridin A induces mitochondrial damage and autophagy in pancreatic cancer and reduces the stability of SMAD4 in Panc-1 cells.

BackgroundPancreatic cancer (PC) is a highly metastatic and lethal cancer with a very low overall 5-year survival rate. There is an urgent need for identifying new therapeutic agents for this deadly disease. Cardiac glycosides (CGs) have been traditionally used for their potent cardiovascular activities and have also recently been reported to exhibit anti-tumor effects. Proscillaridin A (Pro A), a natural CG, has been shown to display anti-tumor effects on multiple cancer types.MethodsThe cytotoxic effect of Pro A on PC cells was determined using cell viability assay, colony formation assay and transwell assay in vitro. Cell apoptosis, cell cycle, reactive oxygen species (ROS) generation, intracellular Ca2+ levels and mitochondrial membrane potential (MMP) were assayed by flow cytometry. Panc-1-xenografted mice model was used to evaluate Pro A’s effect in tumor growth. Mitochondria morphology was observed by transmission electron microscopy. LC3 aggregation was assessed by GFP-LC3 fluorescence microscopy. Gene expression was assayed by western blot or real-time quantitative polymerase chain reaction (qPCR).ResultsPro A inhibits the proliferation, migration and invasion of Panc-1, BxPC-3 and AsPC-1 PC cells in vitro, and Panc-1 cells display the highest sensitivity with an IC50 at the nano-molar level. In vivo, Pro A treatment inhibits tumor progression in Panc-1 xenograft nude mice. Pro A treatment promotes both cell apoptosis and autophagy, and Pro A-treated PC cells display characteristics of mitochondrial damage including increased ROS generation, intracellular Ca2+ levels and disruption of MMP. In addition, high sensitivity towards Pro A of Panc-1 cells compared to BxPC-3 and AsPC-1 cells could be partially attributed to the loss of endogenous SMAD4 expression in the latter.ConclusionsOur findings suggest that Pro A constitutes a promising therapeutic candidate for certain types of PC.

Periplocin Induces Apoptosis of Pancreatic Cancer Cells through Autophagy via the AMPK/mTOR Pathway.

Periplocin, a natural compound, has been shown to induce apoptosis in a variety of cancer cells. However, no research has been conducted to demonstrate that Periplocin has a regulatory effect on autophagy. This study is aimed to determine the effect of Periplocin treatment on autophagy in human pancreatic cancer cells, as well as the underlying mechanisms. Pancreatic cancer cells were treated with different concentrations of Periplocin, and real-time cell analysis (RTCA), colony formation assay, and Ki67 immunofluorescence detection were used to determine cell proliferation. Autophagy protein was detected by immunofluorescence and western blotting. Western blotting was also used to detect the caspase family of apoptotic proteins. Flow cytometry and TUNEL staining were used to detect cell apoptosis. Following treatment with Periplocin, the expression of autophagy genes was detected using RNA-seq. In vivo examination of the effect of Periplocin on autophagy in pancreatic was performed using a xenograft model. Periplocin inhibits the proliferation of CFPAC1 and PANC1 cells and induces autophagy by regulating the AMPK/mTOR pathway. Using the AMPK inhibitor Compound C(CC), both the Periplocin-induced inhibition of cell proliferation and autophagy activation was reduced, which further verified this conclusion. Periplocin inhibits CFPAC1 xenograft tumor growth in nude mice and increases tumor cell autophagy. Collectively, these results have shown that Periplocin promotes autophagy in human pancreatic cancer cells by regulating the AMPK/mTOR pathway.

Abstract 1397: A phenotypic-based drug discovery approach against tumors with MTAP loss

Abstract The Methylthioadenosine phosphorylase (MTAP) gene is in chromosome 9q21 location, which includes p16/CDKNA and is homozygously deleted in about 15% of all cancers, including a large proportion of gliomas, mesotheliomas and T cell lymphomas. MTAP cleaves methylthioadenosine (MTA), a byproduct of polyamine synthesis, to regenerate methionine and adenine. Several functional genomics screens have identified the downstream genes MAT2A and PRMT5 as synthetically lethal with MTAP loss. These two targets have been the subject of several drug discovery efforts. Our approach was based on a forward chemical genetic approach, in which we employed two isogenic cell lines, PANC1, with MTAP loss and PAM cells where MTAP is re-introduced. A library of over 200,000 compounds was screened for hits that affected the viability of PANC1 cell, whereas spared PAM cells. One hit series was found to reproducibly inhibit the proliferation of PANC1 cells with EC50s in the low single digit uM range and one order of selectivity over PAM cells. Phenotype-based optimization resulted in improved analogs, with prototype MTB-22252 exhibiting an EC50 of 47nM in PANC1 cells and 15-fold selectivity. An affinity probe was designed based on an analog of MTB-22252 bearing a biotin moiety via a linker and an azide group for photoactivatable cross-linking with potential interacting proteins. This probe maintained cellular activity and selectivity and it was used in an MS-proteomics experiment in PANC1 cells. The top protein identified by this experiment showed high enrichment over DMSO control and was effectively competed out by MTB-22252. MTB2252 and analogs thermo-stabilized this protein by up to 50C in PANC1 cells. PANC1 cells expressed high levels of this protein, whereas insensitive PAM cell did not express it at all. In additional cell line pairs, sensitivity to MTB-2252 correlated well with the expression levels of this protein. Reintroduction of MTAP in cells lacking MTAP and this new protein, resulted in loss of expression of the new protein, suggesting a regulatory connection. In a wider cell panel of over 130 cell lines, there was correlation of sensitivity with MTAP loss and even better correlation with the expression levels of this protein. Some of the most sensitive cell lines included several colorectal, glioma and hematologic cancer cell lines. In vivo evaluation of MTB-9655 against some of the most sensitive tumor cell lines is on-going. Citation Format: Andreas Goutopoulos, Iris Amanati, Avital Hay-Koren, Ali Fattaey, Philippe Nakache, Dimitri Kovalerchik, Dikla Paz, Simone Botti, Hanna Oppenheimer, Omri Erez, Irit Fainer. A phenotypic-based drug discovery approach against tumors with MTAP loss [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1397.

Thrombospondin-2 acts as a bridge between tumor extracellular matrix and immune infiltration in pancreatic and stomach adenocarcinomas: an integrative pan-cancer analysis

BackgroundThrombospondin-2 (THBS2) is a versatile glycoprotein that regulates numerous biological functions, including the apoptosis-proliferation balance in endothelial cells, and it has been linked to tumor angiogenesis. However, the exact role of THBS2 in human cancer remains unknown. This study aimed to determine THBS2 expression in a pan-cancer analysis and its association with pan-cancer prognosis and to further identify its possible roles in tumor immunity and the extracellular matrix (ECM).MethodsData on THBS2 expression in cancers and normal tissues were downloaded from the Genotype-Tissue Expression portal and UCSC Xena visual exploration tool and analyzed using the ONCOMINE database, Perl programming language, and Gene Expression Profiling and Interactive Analyses vision 2 webserver. In addition, survival prognosis was analyzed using the survival, survminer, limma, and forestplot packages in R v. 4.0.3.Immune and matrix components were also analyzed using R v. 4.0.3. Most importantly, we partially validated the role and mechanism of THBS2 in pancreatic and gastric cancers in vitro using PANC1 and BGC-823 cell lines.ResultsTHBS2 was significantly overexpressed in 17 of the 33 investigated cancers and linked to a poor prognosis in pan-cancer survival analysis. High THBS2 expression was an independent unfavorable prognostic factor in kidney renal papillary cell, mesothelioma, and stomach and pancreatic adenocarcinomas. Immune infiltration and THBS2 expression were also related. THBS2 expression has been linked to immune and stromal scores and immune checkpoint markers in various cancers. The protein–protein interaction network revealed that THBS2 is associated with multiple ECM and immune proteins. THBS2 knockdown decreased the expression of CD47 and matrix metallopeptidase 2 (MMP-2) as well as the proliferation, migration, and invasion of PANC1 and BGC-823 cells in vitro.ConclusionsOur findings suggested that THBS2 might promote cancer progression by remodeling the tumor microenvironment, affecting CD47-mediated signaling pathways, activating the pro-tumor functions of a disintegrin and metalloproteinase with thrombospondin motifs, and enhancing MMP-2 expression. Furthermore, it functions as a bridge between the ECM and immune infiltration in cancer and serves as a potential prognostic biomarker for several cancers, especially pancreatic and gastric adenocarcinomas.

Human amniotic fluid mesenchymal stem cells attenuate pancreatic cancer cell proliferation and tumor growth in an orthotopic xenograft mouse model

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a malignant cancer and chemotherapy ineffectively treats PDAC, leading to the requirement for alternative tumor-targeted treatment. Human amniotic fluid mesenchymal stem cells (hAFMSCs) have been revealed to suppress tumor growth in various cancers and they are a strong candidate for treating PDAC.MethodsTo evaluate the effects of hAFMSCs on human pancreatic carcinoma cells (PANC1, AsPC1 and BxPC3 cell lines) and the possible mechanism involved, an in vitro cell coculture system was used. A PANC1 orthotopic xenograft mouse model was established and hAFMSCs were injected intravenously at 4 weeks post-xenograft.ResultsAn in vitro coculture assay showed that hAFMSCs inhibited PANC1 cell proliferation by inducing S phase cell cycle arrest and increased cell apoptosis in a time-dependent manner. In PANC1 cells, hAFMSCs caused the downregulation of Cyclin A and Cyclin B1 as well as the upregulation of p21 (CDKN1A) at 24 h post coculture. The upregulation of pro-apoptotic factors Caspase-3/-8 and Bax at 24 h post coculture reduced the migration and invasion ability of PANC1 cells through inhibiting the epithelial-mesenchymal transition (EMT) process. In a PANC1 orthotopic xenograft mouse model, a single injection of hAFMSCs showed significant tumor growth inhibition with evidence of the modulation of cell cycle and pro-apoptotic regulatory genes and various genes involved in matrix metallopeptidase 7 (MMP7) signaling-triggered EMT process. Histopathological staining showed lower Ki67 levels in tumors from hAFMSCs-treated mice.ConclusionsOur data demonstrated that hAFMSCs strongly inhibit PDAC cell proliferation, tumor growth and invasion, possibly by altering cell cycle arrest and MMP7 signaling-triggered EMT.

Design of New Potent and Selective Thiophene-Based KV1.3 Inhibitors and Their Potential for Anticancer Activity.

Simple SummaryIn this article, we describe the discovery of a new class of potent and selective thiophene-based inhibitors of the voltage-gated potassium channel KV1.3 and their potential to induce apoptosis and inhibit proliferation. The KV1.3 channel has only recently emerged as a molecular target in cancer therapy. The most potent KV1.3 inhibitor 44 had an IC50 KV1.3 value of 470 nM (oocytes) and 950 nM (Ltk− cells) and appropriate selectivity for other KV channels. New KV1.3 inhibitors significantly inhibited proliferation of Panc-1 cells and KV1.3 inhibitor 4 induced significant apoptosis in tumor spheroids of Colo-357 cells.The voltage-gated potassium channel KV1.3 has been recognized as a tumor marker and represents a promising new target for the discovery of new anticancer drugs. We designed a novel structural class of KV1.3 inhibitors through structural optimization of benzamide-based hit compounds and structure-activity relationship studies. The potency and selectivity of the new KV1.3 inhibitors were investigated using whole-cell patch- and voltage-clamp experiments. 2D and 3D cell models were used to determine antiproliferative activity. Structural optimization resulted in the most potent and selective KV1.3 inhibitor 44 in the series with an IC50 value of 470 nM in oocytes and 950 nM in Ltk− cells. KV1.3 inhibitor 4 induced significant apoptosis in Colo-357 spheroids, while 14, 37, 43, and 44 significantly inhibited Panc-1 proliferation.

Silencing of Carboxypeptidase E expression inhibits proliferation and invasion of Panc-1 pancreatic cancer cells.

Background: Pancreatic cancer is one of the leading cause of cancer-related death globally. The molecular basis of this disease is complex and not fully understood. Previous studies have indicated that carboxypeptidase E (CPE) plays a role in promoting tumorigenesis in many cancer types. Here we have investigated the effect of carboxypeptidase E (CPE), including its isoform, in regulating the proliferation, migration and invasion of Panc-1 cells, a pancreatic cell line. Methods: Panc-1 cells were transfected with CPE siRNA which targets both CPE-wild type and its isoform, or scrambled siRNA, for 24 h and then assayed for proliferation by the MTT and colony formation assays, and migration and invasion by wound healing and matrigel assays, respectively. Results: CPE siRNA treatment of Panc-1 cells down-regulated the expression of CPE mRNA by 94.8%. Silencing of CPE mRNA expression resulted in a significant decrease in proliferation as revealed by the MTT assay and a 62.8% decrease in colony formation. Western blot analysis of expression of Cyclin D1 in Panc-1 cells treated with CPE siRNA showed a decrease of 32.5% compared to scr siRNA treated cells, indicating that CPE regulates proliferation through modulating this cell cycle protein. Additionally, suppression of CPE expression in Panc-1 cells significantly decreased migration and invasion. Conclusions: Our findings indicate that CPE may play an important role in regulating cell proliferation, migration and invasion to promote pancreatic cancer tumorigenesis.

Engineering of a Microscale Niche for Pancreatic Tumor Cells Using Bioactive Film Coatings Combined with 3D-Architectured Scaffolds.

Two-photon polymerization has recently emerged as a promising technique to fabricate scaffolds for three-dimensional (3D) cell culture and tissue engineering. Here, we combined 3D-printed microscale scaffolds fabricated using two-photon polymerization with a bioactive layer-by-layer film coating. This bioactive coating consists of hyaluronic acid and poly(l-lysine) of controlled stiffness, loaded with fibronectin and bone morphogenic proteins 2 and 4 (BMP2 and BMP4) as matrix-bound proteins. Planar films were prepared using a liquid handling robot directly in 96-well plates to perform high-content studies of cellular processes, especially cell adhesion, proliferation, and BMP-induced signaling. The behaviors of two human pancreatic cell lines PANC1 (immortalized) and PAN092 (patient-derived cell line) were systematically compared and revealed important context-specific cell responses, notably in response to film stiffness and matrix-bound BMPs (bBMPs). Fibronectin significantly increased cell adhesion, spreading, and proliferation for both cell types on soft and stiff films; BMP2 increased cell adhesion and inhibited proliferation of PANC1 cells and PAN092 on soft films. BMP4 enhanced cell adhesion and proliferation of PANC1 and showed a bipolar effect on PAN092. Importantly, PANC1 exhibited a strong dose-dependent BMP response, notably for bBMP2, while PAN092 was insensitive to BMPs. Finally, we proved that it is possible to combine a microscale 3D Ormocomp scaffold fabricated using the two-photon polymerization technique with the bioactive film coating to form a microscale tumor tissue and mimic the early stages of metastatic cancer.

Identification of a Pyroptosis-Related Gene Signature and Effect of Silencing the CHMP4C and CASP4 in Pancreatic Adenocarcinoma.

BackgroundPancreatic adenocarcinoma (PAAD) is a highly malignant tumor with an extremely poor prognosis. Pyroptosis has been demonstrated to play an important role in tumor prognosis. However, the expression of pyroptosis-related genes in PAAD and their correlations with prognosis remains unclear.MethodsIn this study, the 36 pyroptosis-related genes that were differentially expressed between normal pancreatic tissues and PAAD tissues were identified via the “limma” R package. Based on these differentially expressed genes (DEGs), a five-gene signature was established by applying the least absolute shrinkage and selection operator Cox regression in the TCGA cohort and was validated in the GEO cohort. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses of DEGs based on the risk model indicated that immune-associated biological processes and pathways were enriched. In vivo, we detected the expressions of CASP4 and CHMP4C by immunohistochemistry in tumor tissues and adjacent normal tissues. In vitro, we silenced CASP4 and CHMP4C to explore their effects on pancreatic cancer cells.ResultsPAAD patients in the low-risk group showed significantly higher survival possibilities than those in the high-risk group. The expressions of CASP4 and CHMP4C in tumor tissue were higher than those in the adjacent normal tissues in vivo. The knockdown of CASP4 significantly inhibited the invasion and migration but not the proliferation of PANC-1 cells. The knockdown of CHMP4C obviously inhibited the proliferation, migration, and invasion of PANC-1 cells.ConclusionPyroptosis-related genes play important roles in predicting the prognosis of PAAD, and CASP4 and CHMP4C affect the metastasis of PAAD.

Silencing of Carboxypeptidase E expression inhibits proliferation and invasion of Panc-1 pancreatic cancer cells

Background: Pancreatic cancer is one of the leading cause of cancer-related death globally. The molecular basis of this disease is complex and not fully understood. Previous studies have indicated that carboxypeptidase E (CPE) plays a role in promoting tumorigenesis in many cancer types. Here we have investigated the effect of carboxypeptidase E (CPE), including its isoform, in regulating the proliferation, migration and invasion of Panc-1 cells, a pancreatic cell line. Methods: Panc-1 cells were transfected with CPE siRNA which targets both CPE-wild type and its isoform, or scrambled siRNA, for 24 h and then assayed for proliferation by the MTT and colony formation assays, and migration and invasion by wound healing and matrigel assays, respectively. Results: CPE siRNA treatment of Panc-1 cells down-regulated the expression of CPE mRNA by 94.8%. Silencing of CPE mRNA expression resulted in a significant decrease in proliferation as revealed by the MTT assay and a 62.8% decrease in colony formation. Western blot analysis of expression of Cyclin D1 in Panc-1 cells treated with CPE siRNA showed a decrease of 32.5% compared to scr siRNA treated cells, indicating that CPE regulates proliferation through modulating this cell cycle protein. Additionally, suppression of CPE expression in Panc-1 cells significantly decreased migration and invasion. Conclusions: Our findings indicate that CPE may play an important role in regulating cell proliferation, migration and invasion to promote pancreatic cancer tumorigenesis.

Peptides against Low Density Lipoprotein (LDL) Aggregation Inhibit Intracellular Cholesteryl Ester Loading and Proliferation of Pancreatic Tumor Cells.

Simple SummaryDyslipidemia is a modifiable risk factor for pancreatic ductal adenocarcinoma (PDAC), one of the most lethal cancers. A key component of dyslipidemia is a high level of small and dense low-density lipoproteins (LDLs). These LDLs have a high probability to be entrapped and modified (aggregated) in the extracellular matrix (ECM), becoming a source of cholesterol for tumor cells. However, the effect of aggregated LDLs on tumor progression has been unexplored. The aim of this work was to determine the effect of modified LDLs on intracellular cholesteryl ester/free cholesterol ratio (CE/FC) and cancer cell growth, and the efficacy of peptides designed to inhibit LDL aggregation on these processes. Our results show that aggregated LDL upregulates the intracellular CE/FC ratio and cell growth in pancreatic cancer and that these upregulatory effects were blocked by peptides against LDL aggregation. We propose that anti-LDL aggregation peptides deserve to be further investigated as anti-tumoral strategies.Dyslipidemia, metabolic disorders and/or obesity are postulated as risk factors for pancreatic ductal adenocarcinoma (PDAC). The majority of patients with these metabolic alterations have low density lipoproteins (LDLs) with increased susceptibility to become aggregated in the extracellular matrix (ECM). LDL aggregation can be efficiently inhibited by low-density lipoprotein receptor-related protein 1 (LRP1)-based peptides. The objectives of this work were: (i) to determine if aggregated LDLs affect the intracellular cholesteryl ester (CE)/free cholesterol (FC) ratio and/or the tumor pancreatic cell proliferation, using sphingomyelinase-modified LDL particles (Aggregated LDL, AgLDL); and (ii) to test whether LRP1-based peptides, highly efficient against LDL aggregation, can interfere in these processes. For this, we exposed human pancreatic cancer cell lines (PANC-1, RWP-1 and Capan-1) to native (nLDL) or AgLDLs in the absence or presence of LRP1-based peptides (DP3) or irrelevant peptides (IP321). Results of thin-layer chromatography (TLC) following lipid extraction indicate that AgLDLs induce a higher intracellular CE/FC ratio than nLDL, and that DP3 but not IP321 counteracts this effect. AgLDLs also increase PANC-1 cell proliferation, which is inhibited by the DP3 peptide. Our results indicate that AgLDL-induced intracellular CE accumulation plays a crucial role in the proliferation of pancreatic tumor cell lines. Peptides with anti-LDL aggregation properties may thus exhibit anti-tumor effects.

Long non-coding RNA DSCAM-AS1 promotes pancreatic cancer progression via regulating the miR-136-5p/PBX3 axis

ABSTRACT LncRNA down syndrome cell adhesion molecule antisense 1 (DSCAM-AS1) plays an important role in tumor progression, but its function in pancreatic cancer is unknown. DSCAM-AS1 level was evaluated by in situ hybridization (ISH) assay and qRT-PCR. DSCAM-AS1 was knocked down in pancreatic cancer cells, and its impacts on cell proliferation, invasion, and migration were detected. The binding relationship among DSCAM-AS1, miR-136-5p, and pre-B-cell leukemia homeobox 3 (PBX3) was investigated by bioinformatic analysis and luciferase reporter assay. An in vivo animal model was constructed to determine the role of DSCAM-AS1 in tumor growth. Our results showed that DSCAM-AS1 was elevated in tumor tissues of pancreatic cancer patients and cell lines. DSCAM-AS1 knockdown efficiently inhibited PANC-1 cell proliferation, migration, and invasion and suppressed tumor growth. DSCAM-AS1 could promote PBX3 expression by sponging miR-136-5p, and its function in pancreatic cancer was partially mediated by the miR-136-5p/PBX3 axis. Overall, DSCAM-AS1 knockdown inhibits pancreatic cancer progression by modulating the miR-136-5p/PBX3 axis.

Acidic Tumor Microenvironment Promotes Pancreatic Cancer through miR-451a/MEF2D Axis.

Pancreatic cancer (PC), as a highly malignant and aggressive solid tumor, is common in the digestive system. The acidic microenvironment is one of the critical markers of cancer. Nonetheless, there are few studies on how the acidic microenvironment affects the development of PC. This study focused on investigating the specific molecular mechanisms of the acidic microenvironment in PC. In our study, qRT-PCR was conducted for examining microRNA (miR)-451a and myocyte enhancer factor 2D (MEF2D) expressions in PANC-1 cells. Then, detailed functional effects of an acidic environment on miR-451a and MEF2D in PANC-1 cells were detected by CCK-8, colony formation, flow cytometry, wound healing, transwell, mitochondrial functionality measurement, JC-1 staining, DCFH-DA staining, and sphere formation assays. The relationship between miR-451a and MEF2D was confirmed by luciferase reporter analysis. Under acidic conditions, the increase of proliferation, migration, and invasion of PANC-1 cells was observed. Moreover, the mitochondrial oxidative respiration-related gene miR-451a was reduced in acidic conditions. In addition, we found that, in PANC-1 cells under an acidic environment, miR-451a overexpression enhanced oxygen consumption, mitochondrial membrane potential (MMP) loss, and ROS generation and inhibited proliferation, migration, invasion, and stemness via sponging MEF2D. In a word, our results revealed that the acidic microenvironment regulated PC progression by affecting the miR-451a/MEF2D axis, indicating a novel avenue for the future treatment of PC.

Gemcitabine in combination with epibrassinolide enhanced the apoptotic response in an ER stress-dependent manner and reduced the epithelial-mesenchymal transition in pancreatic cancer cells.

Gemcitabine is a broad-spectrum antimetabolite and a deoxycytidine analog recognized as a standard therapy alone or in combination with other antineoplastic agents in the therapy of pancreas cancer. Drug resistance following gemcitabine treatment is a common phenomenon; therefore, combinational therapy models are usually preferred. Pancreatic ductal adenocarcinoma, or pancreas cancer, is the fourth leading cause of cancer-related deaths worldwide. With the increasing incidence of pancreatic cancer every year, the mortality rate is also rising significantly because of late diagnosis, and limited chemotherapy options. Adjuvant chemotherapy after surgical resection is the typical option for the treatment of early pancreatic cancer. Mostly, 5-fluorouracil/leucovorin with irinotecan and oxaliplatin (FOLFIRINOX) and gemcitabine/nab-paclitaxel is used for the prognosis of advanced pancreatic cancer; however, chemoresistance usually occurs limiting the effectiveness of the chemotherapy. Therefore, most of the studies are focused on gemcitabine combination with other drugs to overcome the situation. As an apoptotic agent and a member of brassinosteroids, epibrassinolide (EBR) induces endoplasmic reticulum (ER) stress-dependent cell death in different cancer cells, as shown by our group. In this study, we aimed to enhance the gemcitabine apoptotic effect by EBR combined treatment in pancreatic cancer cells. EBR treatment reduced cell viability and inhibited cell proliferation in PANC-1, MIA PaCa-2, and AsPC-1 cells. Each pancreatic cancer cell gave different responses to the EBR treatment because of different aggressiveness. However, EBR induced apoptosis through increasing ROS generation, which was associated with ER stress in PANC-1 and MIA PaCa-2 cells. Gemcitabine alone reduced the cell viability of each pancreatic cancer cell line; however, combination with EBR led to further induction of apoptotic cell death in each pancreatic cancer cell line. In addition, combined treatment of gemcitabine and EBR further decreased N-cadherin and vimentin expressions, suggesting that epithelial-mesenchymal transition of pancreatic cells is reduced. In conclusion, EBR had therapeutic potential to avoid the gemcitabine-induced side effects during the treatment of pancreatic cancer.