Pancreatic Ductal Cancer Cells Research Articles

Adipose tissue-derived extracellular vesicles from obese mice suppressed splenocyte-mediated pancreatic cancer cell death.

Obesity is a risk factor for pancreatic cancer and negatively contributes to the immune system. However, the mechanisms by which obesity mediates these actions are still poorly understood. Recent studies have demonstrated that extracellular vesicles (EVs) are key mediators of communication between cells and may influence various aspects of cancer progression. We aim to explore the influence of EVs derived from adipose tissue of obese mice on cytokine production within the interactions between cancer cells and immune cells. We isolated EVs from the adipose tissue of both C57BL6/J mice and Ob/Ob mice. Subsequently, we treated EVs with Panc02 cells, the murine ductal pancreatic cancer cell line, which were co-cultured with splenocytes. Viability and SMAD4 gene expression were examined in Panc02 cells, and cytokine concentrations of IL-6, IL-4, IL-12, and IL-12p70 were measured in the cultured medium. Interestingly, we observed a significant reduction in splenocyte-mediated Panc02 cell death when treated with EVs derived from the adipose tissue of Ob/Ob mice, compared to those from C57BL6/J mice. Additionally, EVs from Ob/Ob mice-derived adipose tissue significantly increased the levels of IL-4, IL-2, and IL-12p70 in the culture media of Panc02 cells co-cultured with splenocytes, compared to EVs from C57BL6/J mice-derived adipose tissue. Adipose tissue-derived EVs from obese mice suppressed splenocyte-mediated Panc02 cell death and upregulated IL-4, IL-2, and IL-12p70 in cultured medium.

EXPRESSION OF CONCERN: Neurotensin and EGF induce synergistic stimulation of DNA synthesis by increasing the duration of ERK signaling in ductal pancreatic cancer cells.

EXPRESSION OF CONCERN: Neurotensin and EGF induce synergistic stimulation of DNA synthesis by increasing the duration of ERK signaling in ductal pancreatic cancer cells.

Biological activity of recombinant human PDX1 protein produced from Escherichia coli.

Pancreatic and duodenum homeobox 1 (PDX1) is considered as a pivotal transcription factor that acts as a "master regulator" in pancreatogenesis and maintenance of β-cells. Earlier study has reported that PDX1 also functions as a tumor suppressor in human gastric cancer cells by inhibiting cell growth. Here, we report the bioactivity of the purified human PDX1 fusion protein using various assays like cell migration, proliferation, cell cycle analysis, and gene expression. In cancer cells, recombinant PDX1 protein reduced cell migration and proliferation, and arrested cell growth by inducing apoptosis in gastric cancer cells. In pancreatic ductal cancer cells, the application of the PDX1 protein resulted in the induction of insulin gene expression. The results of these experiments demonstrate the biological activity imparted by recombinant human PDX1 fusion protein on gastric and pancreatic cancer cells and its usefulness as a biological tool to elucidate its function in various cellular processes.

Devil's club Falcarinol inhibits human glioblastoma cell proliferation and tumor progression in xenografts

BackgroundEffective treatment for human glioblastoma is lacking and a novel effective therapeutic agent is urgently needed. It is known that falcarinol-type polyacetylenes (FC-type PAs) are bioactive ingredients in ginseng and other plants from the Araliaceae family. Our earlier study showed that polyacetylene Falcarinol (FC), 1,2-dihydrofalcarinol (FCH) and 3-acetoxyfalcarinol (FCA) are potent proliferation inhibitors of human pancreatic ductal cancer cell lines. MethodsIn this study we evaluated the bioactivities of these synthetic FC-type polyacetylenes (FC-type PAs) on human glioblastoma DBTRG-05MG cell line with cell viability, cell proliferation, cell migration, wound healing and clonogenic assays. We further evaluated synthetic FC on inhibition of DBTRG tumor bearing NOD-Prkdcem26Il2rgem26/Nju (NCG) mice. ResultsFC dose dependently shifted DBTRG-05MG cells toward apoptosis as well as necrosis. FC and FCA, but not FCH, at near IC50 concentration inhibited colony formation in a 3-week clonogenic assay. Twice weekly intra-peritoneal injection of FC at 10 mg/kg significantly reduced tumor growth and tumor size compared to untreated control animals. ConclusionsSynthetic FC inhibits human glioblastoma cancer cell proliferation in vitro and in vivo. The availability of synthetic FC and other FC-type PAs in sufficient quantity and consistent quality would facilitate and expedite further research leading to potential clinical applications.

PRSS1 Mutations Affect Pancreatic Ductal Adenocarcinoma Radiosensitivity via AKT and Extracellular Regulated Protein Kinases Pathways

Radioresistance is the leading cause of failed radiation therapy for pancreatic ductal cancer (PDAC). The relevance of the cationic trypsinogen gene (PRSS1) in PDAC radioresistance is unknown, despite its association with tumor responses to therapy in numerous malignancies. Here we established two PRSS1 point mutation PDAC cell lines: c. 338 T > G and c.410 C > T. Compared to their parental cells, elevated AKT and ERK phosphorylation concentrations were observed in Panc-1 and MIA PaCa-2 c. 338 T > G and c.410 C > T cells with point mutations. The PRSS1 mutation restored the sensitivity of radioresistant cells to radiation through increased ionizing radiation-induced apoptosis by down regulating p-AKT and p-ERK. Based on these results, we hypothesized that a PRSS1 mutation in PDAC increased cell radiosensitivity by decreasing p-AKT and p-ERK. Our findings provide a molecular basis for optimizing radiation in patients with PDAC.

Long noncoding RNA negative regulator of antiviral response contributes to pancreatic ductal adenocarcinoma progression via targeting miR-299-3p.

BACKGROUNDPancreatic ductal cancer (PDAC) has high malignancy and poor prognosis. Long noncoding RNAs (lncRNAs) are associated with high levels of malignancy, including PDAC. However, the biological and clinical significance of negative regulator of antiviral response (NRAV) in PDAC is unclear.AIMTo study the regulatory role of lncRNA NRAV in PDAC.METHODSGEPIA analyzed lncRNA NRAV and miRNA (miR-299-3p) expression levels in PDAC tissues and measured them in PDAC cells by quantitative measurements in real time. The specific role of NRAV and miR-299-3p in cell proliferation and transfer potential was evaluated by cell formation analysis, Cell Counting Kit-8 and Transwell analysis. The relationship between NRAV and miR-299-3p was studied by predictive bioinformatics, RNA immunoassay, and fluorescence enzyme analysis. In vivo experiments included transplantation of simulated tumor cells under naked mice.RESULTSThe expression level of lncRNA NRAV was higher in both tumor tissues and cell lines of PDAC and was negatively associated with the clinical survival of PDAC patients. Functionally, overexpression of NRAV promoted cell proliferation and metastasis of PDAC cells, while knockdown of NRAV reversed these effects. Finally, NRAV was performed as a molecular sponge of miR-299-3p. Moreover, overexpression of miR-299-3p could reverse the promoting effects of NRAV on cell proliferation and metastasis of PDAC cells.CONCLUSIONNRAV facilitates progression of PDAC as a molecular sponge of miR-299-3p and may be a potential molecular marker for diagnosis and treatment of PDAC.

Paracrine Signaling From Pancreatic Ductal Cancer Cells Induced Programmed-Cell Death in 3T3-L1 Adipocytes Through Apoptosis and Ferroptosis

ObjectivesCancer cachexia is a prominent feature of patients with pancreatic ductal adenocarcinoma (PDAC). Cachexia is characterized by aberrant inflammatory responses, rapid loss of fat and muscle mass. Emerging evidence revealed that adipose tissue loss precedes muscle loss in those with cancer cachexia, suggesting that blocking cachectic fat loss is a therapeutic target. However, the underlying mechanisms by which cancer cells promote fat loss have not yet been established. We aim to investigate the molecular mechanisms behind the cachectic fat loss in PDAC patients. MethodsThe cell line derived from PDAC KPC mice (KrasG12D; Trp53R172H; Pdx1-Cre) was cultured, and conditioned-medium (CM) was collected for detecting proinflammatory cytokine levels by ELISA. An in vitro model was used to determine paracrine crosstalk between PADC and adipose tissue by transferring conditioned-medium of KPC (KPC-CM) to fully differentiated 3T3-L1 adipocytes. The effects of KPC-CM on lipolysis, lipogenesis- and iron homeostasis-related protein, proinflammatory gene expression, and signaling pathways for programmed cell death were examined. Intracellular free iron concentration and ROS production were also determined by using fluorometric assays. ResultsOur findings showed that CM of KPC cells contains various proinflammatory cytokines and chemokines associated with immune cell infiltration and oxidative stress, including CXCL1, LIX, CCL11, CCL2, IL-12, and TNFa. Addition of KPC-CM to 3T3-L1 adipocytes promoted glycerol release and phosphorylation of hormone-sensitive lipase in a dose-dependent manner, supporting augmented lipolysis. Moreover, KPC-CM reduced the adipocyte-specific gene and protein expression and promoted caspase 3-mediated apoptotic cell death. Concurrently, KPC-CM induced modification of iron metabolism, including the decreased expression of glutathione peroxidase 4 (GPX4) and increased nuclear receptor coactivator 3 (NCOA4), two biomarkers for iron-dependent programmed cell death, ferroptosis. In parallel, KPC-CM increased the intracellular Fe2+ levels and ROS production in 3T3-L1 adipocytes. ConclusionsCollectively, our results revealed that the crosstalk between adipocyte tissue and PDAC CM triggers deregulation of iron homeostasis and lipid metabolism, leading to programmed cell death of adipocytes. Funding SourcesN/A.

COX6B2 drives metabolic reprogramming toward oxidative phosphorylation to promote metastasis in pancreatic ductal cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer type with poor prognosis due to its high metastatic potential, however, the role of metabolic reprogramming in the metastasis of PDAC cell is not known. Here, we report that COX6B2 drive metastasis but not cancer cell proliferation in PDAC by enhancing oxidative phosphorylation function (OXPHOS). Transcriptome and clinical analyses revealed that cytochrome c oxidase subunit 6B2 (COX6B2) positively associated with metastasis of PDAC cells. Knockdown of COX6B2 in PDAC cells tuned down the assembly of complex IV and downregulated the function of OXPHOS, whereas re-expression of COX6B2 restored the function of OXPHOS and metastatic potential. Mechanistically, COX6B2 upregulated OXPHOS function to active purinergic receptor pathway for the metastasis of PDAC cells. Notably, the metastatic potential in PDAC could be reversely regulated by metformin, a drug was found accelerating the degradation of COX6B2 mRNA in this study. Collectively, our findings indicated that a complex metabolic control mechanism might be involved in achieving the balance of metabolic requirements for both growth and metastasis in PDAC, and regulation of the expression of COX6B2 could potentially encompass one of the targets.

Dissecting the single cell profile of pancreatic ductal adenocarcinoma

Background: The interplay of the tumor, stroma and immune system is becoming increasingly recognized as crucial in the pathogenesis of pancreatic ductal adenocarcinoma (PDAC). Traditional bulk RNA sequencing relies on utilizing RNA from a tumor specimen, and subjecting it to modern RNA sequencing techniques. Although this technique has allowed us to make great strides in understanding the heterogeneity and profile of pancreatic adenocarcinoma, we are unable to track RNA expression on the single cell level—which would allow us to pinpoint how treatments or lack thereof are contributing to phenotype and resultant survival. Methods: Immediately following resection of a pancreatic cancer specimen or alternatively after biopsy of a metastatic lesion, a tumor suspension is made. The tumor is broken down with a combination of mechanical and enzymatic digestion. This is followed by isolation of live single cells, and removal of RBCs and debris (Figure 1A). We immediately run a cell suspension of 10,000 live cells on the 10× Genomics single cell platform (Figure 1B), whereby each individual cell receives a unique barcode that it carries throughout the sequencing process. We then perform RNA sequencing. After sequencing, all cells with mitochondrial DNA content >10%, consistent with a dead cell, are removed from further analysis. We are left with a single cell RNA profile of live cells from the tumor. Results: Thus far we have processed 4 primary pancreatic ductal adenocarcinoma specimens, and 1 metastatic lesion, from a total of 5 patients, with 3 samples analyzed thus far. On average, we are left with 2470 live cells per patient sample. For each patient, we were able to identify 12 unique cell populations. Patient 1174, underwent a Whipple without preoperative chemoradiotherapy for a resectable PDAC head mass (T1cN2 on final path). Single cell analysis of the tumor specimen, identified 42.7% of the tumor mass to be PDAC cells (Figure 1C). In this case 4 distinct tumor subpopulations can be identified (Figure 1C). T and B lymphocytes comprised 35.6% of infiltrating cells, macrophages comprised 13.2% of the tumor population, with mast cells, pancreatic stellate cells, acinar cells and endothelial cells comprising the remainder of the tumor mass. Each cell population is identified by multiple characteristic markers, with 1 identifying marker displayed in Figure 1C. Example characteristic marker being KRT19 for pancreatic ductal cancer cells. In contrast to patient 1174, patient 1105, who received upfront neoadjuvant chemotherapy, but was found to be unresectable in the operating room, had a liver tumor with only 23.9% PDAC cells, and ∼50% of infiltrating cells comprising T and B lymphocytes, as well as NK cells. Finally, patient 1160, who had a resectable pancreatic head mass (T2N1 on final path) and did not receive preoperative therapy, had 20.6% PDAC cells, ∼33% infiltrating lymphocytes, and 20% macrophages. Conclusion: Single cell RNA sequencing is a viable strategy to better dissect the makeup of PDACs. It allows for RNA profiling on a single cell level, and provides a landscape of the tumor and tumor microenvironment in a non-biased fashion. Our group is enrolling multiple patients treated and not treated prior to pancreatic cancer resection, as well as consecutive specimens (pre treatment and post surgical) from several patients in order to better assess both the PDAC landscape and how it changes based on different factors such as chemotherapy, stage or location.

Oncogenic HSP60 regulates mitochondrial oxidative phosphorylation to support Erk1/2 activation during pancreatic cancer cell growth

HSP60 is a mitochondrial localized quality control protein responsible for maintaining mitochondrial function. Although HSP60 is considered both a tumor suppressor and promoter in different types of cancer, the role of HSP60 in human pancreatic ductal adenocarcinoma (PDAC) remains unknown. In this study, we demonstrated that HSP60 was aberrantly expressed in human pancreatic cancer tissues and cell lines. Analysis of the Cancer Genome Atlas database revealed that HSP60 expression is positively correlated with pancreatic cancer. Further, knockdown of HSP60 attenuated pancreatic ductal cancer cell proliferation and migration/invasion, whereas ectopic expression of HSP60 increased tumorigenesis. Using an in vivo tumorigenicity assay, we confirmed that HSP60 promoted the growth of pancreatic ductal cancer cells. Functional analyses demonstrated that HSP60 plays a key role in the regulation of mitochondrial function. Mechanistically, both HSP60 knockdown and oxidative phosphorylation (OXPHOS) inhibition by metformin decreased Erk1/2 phosphorylation and induced apoptosis and cell cycle arrest, whereas Erk1/2 reactivation with EGF promoted cell proliferation. Intriguingly, in vitro ATP supplementation partially restored Erk1/2 phosphorylation and promoted proliferation in PDAC cells with HSP60 knockdown and OXPHOS inhibition. These results suggest that mitochondrial ATP is an important sensor of Erk1/2 regulated apoptosis and the cell cycle in PDAC cells. Thus, our findings indicate for the first time that HSP60 may serve as a novel diagnostic target of human pancreatic cancer, and that inhibition of mitochondrial function using drugs such as metformin may be a beneficial therapeutic strategy targeting pancreatic cancer cells with aberrant function of the HSP60/OXPHOS/Erk1/2 phosphorylation axis.

Bit1 mediates the malignant behaviors in pancreatic cancer and its potential clinical significance

Objective: To investigate the potential role of Bit1 in the pathogenesis of pancreatic ductal cancer cells(PDAC) and its potential clinical application value. Methods: Real-time PCR and Western blot were employed to detect the expression of Bit1 in six pancreatic cancer cells, then the tool cells were selected to further study the function of Bit1.PolyHEMA was used to monitor the suspended cell culture condition in vitro.The invasion and migration abilities of pancreatic cancer cells were detected through Transwell assay. Western blot and confocal assay were used to explore the potential mechanism of Bit1 in the process of metastasis.The expression of Bit1 was detected through tissue microarray, the potential relationship between Bit1 and other clinical factors were analyzed. Results: The results of real-time PCR and Western blot indicated that the expression of Bit1 was highest in the PANC1 cells and lowest in the Mia paca2 cells (gene: 3.13±0.40 vs. 1.00±0.35, protein: 1.77±1.00 vs. 0.23±0.45). The shBit1 PANC1 and Bit1-OE(over expression) Mia paca2 cells were successfully constructed.Bit1 over expression could promote the anoikis rate of Mia paca2 cells, and Bit knockdown could inhibit the anoikis incidence.Bit1 over expression suppressed the motility and invasion of Mia paca2 cells, but Bit1 knockdown could accelerate the migration and invasion ability of PANC1 cells.Bit1 could potentially affect pancreatic cancer cells' malignant behaviors through epithelial-mesenchymal transition process.Bit1 expression was significantly associated with pancreatic cancer's neural invasion (P<0.05). Conclusions: Bit1 could affect the anoikis incidence of pancreatic cancer, Bit1 negatively affect the migration and invasion abilities of PDAC, the EMT process was potentially involved in the whole modulation process.Bit1 expression is associated with neural invasion in pancreatic cancer patients.

Abstract A41: GPRC5A acts as a potent oncogene in pancreatic cancer

Abstract Background: GPRC5A is an orphan G-protein coupled receptor acting as an oncogene in some cancers and as a tumor suppressor in other. Its role in pancreatic cancer has not been studied systematically. ELAV-like protein 1, also known as “HuR,” is an RNA binding protein that is encoded by the ELAVL1 gene. HuR post-transcriptionally regulates multiple genes, plays key roles in human malignancies, and is known to mediate the efficacy of gemcitabine. Our preliminary data indicated that, in pancreatic cells, GPRC5A transcription responded to gemcitabine treatment and that its mRNA likely interacted with HuR. Methods: We immunohistochemically analyzed GPRC5A protein expression in primary and metastatic pancreatic cancer samples. We examined the effects of GPRC5A on pancreatic cells’ ability to proliferate, migrate, and invade, and how gemcitabine modulates this ability. We also studied the interaction between GPRC5A and HuR and examined how it is affected by gemcitabine. Lastly, we used microarray expression analyses to identify the genes and pathways linked to GPRC5A in pancreatic cancer cells. Results: We analyzed public mRNA expression data from 675 human cancer cell lines and 10,609 samples from The Cancer Genome Atlas (TCGA): GPRC5A mRNA abundance in pancreatic cancer was the highest (cell lines) or second highest (TCGA) among all tissues and cancer types. We analyzed an independent set of 252 pancreatic normal and cancer samples: we found GPRC5A mRNA to be up-regulated in primary tumor samples compared to normal pancreas, and even further up-regulated in metastatic pancreatic cancer. We immunostained 208 cores (103 primary and metastatic pancreatic cancer samples): we found GPRC5A protein to be barely expressed in normal pancreatic ductal cells and highly expressed in both primary and metastatic pancreatic ductal cancer cells. We carried out in vitro studies of multiple pancreatic cancer cell lines: we found that increasing GPRC5A protein levels promoted pancreatic cancer cell growth and migration whereas siRNA-mediated knockdown of GPRC5A impaired proliferation and invasion. We found that gemcitabine treatment enhanced GPRC5A expression in pancreatic cancer cells. Conversely, siRNA-mediated knockdown of GPRC5A sensitized the cells to gemcitabine. We showed that HuR/ELAVL1 up-regulated GPRC5A protein in the first 18 hours following gemcitabine treatment by stabilizing GPRC5A’s mRNA through binding to at least on site in GPRC5A’s 3´UTR. Microarray analysis showed that GPRC5A overexpression impacts several signaling networks. Conclusions: Our results indicate that GPRC5A acts as an oncogene in pancreatic cancer. Unexpectedly, gemcitabine was found to increase GPRC5A's mRNA and protein levels. We showed that this increase is mediated by HuR, a known enabler of gemcitabine efficacy, through a direct interaction between HuR and GPRC5A’s mRNA. It appears that the sensitivity of pancreatic cancer cells to gemcitabine can be augmented through down-regulation of GPRC5A. Citation Format: Honglei Zhou, Aristeidis Telonis, Yi Jing, Masaya Jimbo, Fernando Blanco, Eric Londin, Jonathan Brody, Isidore Rigoutsos.{Authors}. GPRC5A acts as a potent oncogene in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A41.

Identification of drugs that restore primary cilium expression in cancer cells.

The development of cancer is often accompanied by a loss of the primary cilium, a microtubule-based cellular protrusion that functions as a cellular antenna and that puts a break on cell proliferation. Hence, restoration of the primary cilium in cancer cells may represent a novel promising approach to attenuate tumor growth. Using a high content analysis-based approach we screened a library of clinically evaluated compounds and marketed drugs for their ability to restore primary cilium expression in pancreatic ductal cancer cells. A diverse set of 118 compounds stimulating cilium expression was identified. These included glucocorticoids, fibrates and other nuclear receptor modulators, neurotransmitter regulators, ion channel modulators, tyrosine kinase inhibitors, DNA gyrase/topoisomerase inhibitors, antibacterial compounds, protein inhibitors, microtubule modulators, and COX inhibitors. Certain compounds also dramatically affected the length of the cilium. For a selection of compounds (Clofibrate, Gefitinib, Sirolimus, Imexon and Dexamethasone) their ability to restore ciliogenesis was confirmed in a panel of human cancer cell line models representing different cancer types (pancreas, lung, kidney, breast). Most compounds attenuated cell proliferation, at least in part through induction of the primary cilium, as demonstrated by cilium removal using chloral hydrate. These findings reveal that several commonly used drugs restore ciliogenesis in cancer cells, and warrant further investigation of their antineoplastic properties.

The Dipeptide Monoester Prodrugs of Floxuridine and Gemcitabine—Feasibility of Orally Administrable Nucleoside Analogs

Dipeptide monoester prodrugs of floxuridine and gemcitabine were synthesized. Their chemical stability in buffers, enzymatic stability in cell homogenates, permeability in mouse intestinal membrane along with drug concentration in mouse plasma, and anti-proliferative activity in cancer cells were determined and compared to their parent drugs. Floxuridine prodrug was more enzymatically stable than floxuridine and the degradation from prodrug to parent drug works as the rate-limiting step. On the other hand, gemcitabine prodrug was less enzymatically stable than gemcitabine. Those dipeptide monoester prodrugs exhibited 2.4- to 48.7-fold higher uptake than their parent drugs in Caco-2, Panc-1, and AsPC-1 cells. Floxuridine and gemcitabine prodrugs showed superior permeability in mouse jejunum to their parent drugs and exhibited the higher drug concentration in plasma after in situ mouse perfusion. Cell proliferation assays in ductal pancreatic cancer cells, AsPC-1 and Panc-1, indicated that dipeptide prodrugs of floxuridine and gemcitabine were more potent than their parent drugs. The enhanced potency of nucleoside analogs was attributed to their improved membrane permeability. The prodrug forms of 5′-l-phenylalanyl-l-tyrosyl-floxuridine and 5′-l-phenylalanyl-l-tyrosyl-gemcitabine appeared in mouse plasma after the permeation of intestinal membrane and the first-pass effect, suggesting their potential for the development of oral dosage form for anti-cancer agents.

Vitamin E δ-Tocotrienol Induces p27Kip1-Dependent Cell-Cycle Arrest in Pancreatic Cancer Cells via an E2F-1-Dependent Mechanism

Vitamin E δ-tocotrienol has been shown to have antitumor activity, but the precise molecular mechanism by which it inhibits the proliferation of cancer cells remains unclear. Here, we demonstrated that δ-tocotrienol exerted significant cell growth inhibition pancreatic ductal cancer (PDCA) cells without affecting normal human pancreatic ductal epithelial cell growth. We also showed that δ-tocotrienol-induced growth inhibition occurred concomitantly with G1 cell-cycle arrest and increased p27Kip1 nuclear accumulation. This finding is significant considering that loss of nuclear p27Kip1 expression is a well-established adverse prognostic factor in PDCA. Furthermore, δ-tocotrienol inactivated RAF-MEK-ERK signaling, a pathway known to suppress p27Kip1 expression. To determine whether p27Kip1 induction is required for δ-tocotrienol inhibition of PDCA cell proliferation, we stably silenced the CDKN1B gene, encoding p27Kip1, in MIAPaCa-2 PDCA cells and demonstrated that p27Kip1 silencing suppressed cell-cycle arrest induced by δ-tocotrienol. Furthermore, δ-tocotrienol induced p27Kip1 mRNA expression but not its protein degradation. p27Kip1 gene promoter activity was induced by δ-tocotrienol through the promoter's E2F-1 binding site, and this activity was attenuated by E2F-1 depletion using E2F-1 small interfering RNA. Finally, decreased proliferation, mediated by Ki67 and p27Kip1 expression by δ-tocotrienol, was confirmed in vivo in a nude mouse xenograft pancreatic cancer model. Our findings reveal a new mechanism, dependent on p27Kip1 induction, by which δ-tocotrienol can inhibit proliferation in PDCA cells, providing a new rationale for p27Kip1 as a biomarker for δ-tocotrienol efficacy in pancreatic cancer prevention and therapy.

DCB-3503, a Tylophorine Analog, Inhibits Protein Synthesis through a Novel Mechanism

BackgroundDCB-3503, a tylophorine analog, inhibits the growth of PANC-1 (human pancreatic ductal cancer cell line) and HepG2 (human hepatocellular cancer cell line) tumor xenografts in nude mice. The inhibition of growth leads to cancer cell differentiation instead of cell death. However, the mechanisms of action of tylophorine analogs is unknown.Methodology/Principal FindingsIn this study, we show that DCB-3503 suppresses the expression of pro-oncogenic or pro-survival proteins with short half-lives, including cyclin D1, survivin, β-catenin, p53, and p21, without decreasing their mRNA levels. Proteasome inhibitor reversed the inhibitory effect of DCB-3503 on expression of these proteins. DCB-3503 inhibited the incorporation of radiolabeled amino acid and thymidine, and to a much lesser degree of uridine, in a panel of cell lines. The mechanism of inhibition of protein synthesis is different from that of cycloheximide (CHX) as assayed in cell culture and HeLa in vitro translation system. Furthermore, in contrast to rapamycin, DCB-3503 does not affect protein synthesis through the mTOR pathway. DCB-3503 treatment shifts the sedimentation profiles of ribosomes and mRNAs towards the polysomal fractions while diminishing monosome abundance, indicative of the inhibition of the elongation step of protein synthesis. Preferential down regulation of several studied proteins under these conditions is likely due to the relative short half-lives of these proteins.Conclusion/SignificanceThe inhibitory effect of DCB-3503 on translation is apparently distinct from any of the current anticancer compounds targeting protein synthesis. Translation inhibitors with novel mechanism could complement current chemotherapeutic agents for the treatment of human cancers and suppress the occurrence of drug resistance.

Synergistic effect of trichostatin A and 5‐aza‐2′‐deoxycytidine on growth inhibition of pancreatic endocrine tumour cell lines: A proteomic study

Our research group recently reported that pancreatic endocrine cancer cell lines are sensitive to the HDAC inhibitor trichostatin A (TSA). In the present paper, we show that the combined treatment of pancreatic endocrine tumour cell lines with TSA and the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (DAC) determines a strong synergistic inhibition of proliferation mainly due to apoptotic cell death. Proteomic analysis demonstrates that the modulation of specific proteins correlates with the antiproliferative effect of the drugs. A schematic network clarifies the most important targets or pathways involved in pancreatic endocrine cancer growth inhibition by single or combined drug treatments, which include proteasome, mitochondrial apoptotic pathway and caspase related proteins, p53 and Ras related proteins. A comparison between the patterns of proteins regulated by TSA or DAC in endocrine and ductal pancreatic cancer cell lines is also presented.

Decreased Expression of Angiotensin-Converting Enzyme 2 in Pancreatic Ductal Adenocarcinoma Is Associated with Tumor Progression

Angiotensin II (ANG II), the biologically active peptide of the renin-angiotensin system (RAS), is generated by angiotensin-converting enzyme (ACE) and is a regulator of cardiovascular homeostasis. Recently, there has been increasing evidence that ANG II is involved in the regulation of cell proliferation and migration, as well as angiogenesis via the ANG II-type 1 receptor (AT1R). These findings suggest that the ACE-ANG II-AT1R pathway is related to cancer biology. Previous reports have shown that ACE is preferentially expressed in pancreatic ductal adenocarcinoma (PDAC) tissues. Recently a homologue of ACE, angiotensin-converting enzyme 2 (ACE2), was reported to counterbalance the function of ACE, but the expression and role of ACE2 in PDAC are still unclear. In the present study, we analyzed the expression of ACE2 in invasive human PDAC and surrounding non-malignant tissues by Western blot analysis and immunohistochemistry. The ANG II concentration in homogenates of pancreatic tissues was measured with ELISA, and ACE2 protein was detected by Western blot analysis in BxPC3 and SW1990 human pancreatic ductal cancer cells. We have shown for the first time that the expression of ACE2 is decreased in PDAC tissues, in which ANG II was accumulated. Treatment of BxPC3 and SW1990 cells with ANG II decreased the expression of ACE2. Therefore, ANG II may contribute to the down-regulation of ACE2. Moreover, reduction of ACE2 expression by RNA interference promoted the proliferation of cultured pancreatic cancer cells. These findings suggest that ACE2 may have clinical potential as a novel molecular target for the treatment of PDAC.

Intravital Microscopic Characterization of Suramin Effects in an Orthotopic Immunocompetent Rat Model of Pancreatic Cancer

ObjectivesWe investigated the effect of suramin on tumor growth and spread in an immunocompetent, orthotopic rat model of pancreatic cancer and analyzed the tumor vasculature by intravital microscopy. Methods and MethodsIn vitro, rat ductal pancreatic cancer cells (DSL-6A) were incubated with suramin (10–800 µg/ml), and cell proliferation was assessed. In vivo, DSL-6A tumors were induced in the pancreas of Lewis rats. Animals received suramin (60 mg/kg, weekly i.p.) or the vehicle (controls). Treatment started after 3 days. Intravital microscopy after 1, 4, and 8 weeks quantified diameter, density, and permeability of tumor vessels. Primary tumor volume, local infiltration, and metastatic spread were determined at autopsy. Microvessel density was analyzed by immunohistochemistry. ResultsIn vitro, proliferation was inhibited by suramin up to 95%. In vivo, all controls developed extensive tumor growth and spread. No tumor was detectable in half of the suramin-treated animals after 8 weeks; tumor dissemination was almost completely depressed. Suramin therapy resulted in a complete regression of tumor macrovessels and a significant reduction of microvessel density. ConclusionSuramin significantly reduces primary tumor growth and dissemination in a clinically relevant rat model of pancreatic cancer and seems to play an important role for the inhibition of tumor angiogenesis.

Notch Signaling Is Required for Exocrine Regeneration After Acute Pancreatitis

The mechanisms for tissue regeneration and renewal after acute pancreatitis are not well understood but may involve activation of Notch signaling. To study the effect of Notch signaling ablation during acute experimental pancreatitis, we used a chemical and genetic approach to ablate Notch signaling in cerulein-induced pancreatitis in mice. Acute pancreatitis was induced by cerulein treatment in mice treated with the gamma-secretase inhibitor dibenzazepine or in conditional Notch1 knockout mice. Mice were characterized using immunohistologic, biochemical, and molecular methods. To investigate Notch and beta-catenin interaction, acinar 266-6 cells were analyzed using transfection and biochemical assays. Loss of Notch signaling results in impaired regeneration after acute pancreatitis with fewer mature acinar cells in dibenzazepine-treated and Notch1-deficient mice in the regenerative phase 3 days after induction. beta-catenin expression was increased and prolonged during exocrine regeneration. Crosstalk between Notch and beta-catenin-mediated signaling was identified, with Notch1-IC inhibiting beta-catenin-mediated transcriptional activity. This inhibition was dependent on a functional RAM domain. Inhibition of Notch signaling in vivo leads to impaired regeneration of the exocrine pancreas after acute pancreatitis. Our results suggest an interaction of Notch and Wnt signaling in pancreatic acinar cells, providing evidence for a role of these pathways in the regulation of the maturation process of acinar cells.