Human Pancreatic Ductal Cells Research Articles

PDK4 dictates metabolic resistance to ferroptosis by suppressing pyruvate oxidation and fatty acid synthesis

Although induction of ferroptosis, an iron-dependent form of non-apoptotic cell death, has emerged as an anticancer strategy, the metabolic basis of ferroptotic death remains poorly elucidated. Here, we show that glucose determines the sensitivity of human pancreatic ductal carcinoma cells to ferroptosis induced by pharmacologically inhibiting system xc-. Mechanistically, SLC2A1-mediated glucose uptake promotes glycolysis and, thus, facilitates pyruvate oxidation, fuels the tricyclic acid cycle, and stimulates fatty acid synthesis, which finally facilitates lipid peroxidation-dependent ferroptotic death. Screening of a small interfering RNA (siRNA) library targeting metabolic enzymes leads to identification of pyruvate dehydrogenase kinase 4 (PDK4) as the top gene responsible for ferroptosis resistance. PDK4 inhibits ferroptosis by blocking pyruvate dehydrogenase-dependent pyruvate oxidation. Inhibiting PDK4 enhances the anticancer activity of system xc- inhibitors invitro and in suitable preclinical mouse models (e.g., a high-fat diet diabetes model). These findings reveal metabolic reprogramming as a potential target for overcoming ferroptosis resistance.

Proteome-Wide Alterations of Asymmetric Arginine Dimethylation Associated With Pancreatic Ductal Adenocarcinoma Pathogenesis.

Arginine methylation catalyzed by protein arginine methyltransferases (PRMTs) performs essential roles in regulating cancer initiation and progression, but its implication in pancreatic ductal adenocarcinoma (PDAC) requires further elucidation. In this study, asymmetric dimethylarginine (ADMA)-containing peptides in PDAC cell line PANC-1 were identified by label-free quantitative proteomics combined with affinity purification, using human non-cancerous pancreatic ductal epithelium cell line HPDE6c7 as the control. In total, 289 ADMA sites in 201 proteins were identified in HPDE6c7 and PANC-1 cells, including 82 sites with lower dimethylation and 37 sites with higher dimethylation in PANC-1 cells compared with HPDE6c7 cells. These ADMA-containing peptides demonstrated significant enrichment of glycine and proline residues in both cell lines. Importantly, leucine residues were significantly enriched in ADMA-containing peptides identified only in HPDE6c7 cells or showing lower dimethylation in PANC-1 cells. ADMA-containing proteins were significantly enriched in multiple biological processes and signaling cascades associated with cancer development, such as spliceosome machinery, the Wnt/β-catenin, Hedgehog, tumor growth factor beta (TGF-β), and mitogen-activated protein kinase (MAPK) signaling pathways. Moreover, PDAC cell lines with enhanced cell viability showed lower PRMT4 protein abundance and global ADMA-containing protein levels compared with HPDE6c7. PRMT4 overexpression partially recovered ADMA-containing protein levels and repressed viability in PANC-1 cells. These results revealed significantly altered ADMA-containing protein profiles in human pancreatic carcinoma cells, which provided a basis for elucidating the pathogenic roles of PRMT-mediated protein methylation in pancreatic cancer.

Coxsackievirus-B4 Infection Can Induce the Expression of Human Endogenous Retrovirus W in Primary Cells.

Human Endogenous Retrovirus W Envelope (HERV-W ENV) mRNA or protein can be found in peripheral blood mononuclear cells (PBMCs) and exocrine pancreas of patients with type 1 diabetes (T1D). Further, previous observations have shown an association between enteroviral infection and development of T1D; specifically, coxsackievirus-B (CV-B) has been detected in the blood and pancreas of patients with T1D. Notably, viruses can activate HERV-W expression. Hence, we evaluated the effect of CV-B4 infection on HERV-W ENV mRNA expression. Primary human pancreatic ductal cells were obtained from five brain-dead donors. In the pancreatic cells of three donors, the HERV-W ENV mRNA level measured using RT-qPCR was upregulated upon CV-B4 infection. The HERV-W ENV protein was detected in the infected cells using the immunoblot assay. In human PBMCs inoculated with CV-B4 or when CV-B4 was incubated with an enhancing serum, the HERV-W ENV mRNA level was higher than the background RNA level. In monocyte-derived macrophages obtained from 5 of 13 donors, the HERV-W ENV mRNA level was higher in cultures inoculated with CV-B4 than in the control. Therefore, CV-B4 can upregulate or induce the transcription of a certain HERV-W ENV copy (or copies) in primary cell cultures, such as monocytes, macrophages, and pancreatic cells.

Abstract A32: The small GTPase Rac1 controls the stability of Yes-Associated Protein (YAP) independently of the LATS1/2 kinases

Abstract Activating mutations in the K-Ras gene are the earliest and most common genetic alterations detected in human pancreatic cancer (PC) specimens. In genetically engineered mouse models of PC, oncogenic K-Ras drives the formation of PanIN precursor lesions and their progression to invasive PC. Oncogenic K-Ras signals through at least four effector pathways that play distinct roles in malignant transformation: MAPK pathway, PI3K pathway, Ral-GDS, and the Rac1 GTPase (Ras-related C3 botulinum toxin substrate 1). Among these effectors, the least well understood is the Rac1 pathway. Yet, this pathway has now been shown to be critical for Ras-driven PC development and malignant transformation. However, the specific mechanisms by which Rac1 promotes anchorage-independent growth and tumorigenicity are still unclear. In recent studies, we have discovered that the Rac1 pathway regulates the stability of the Yes-Associated Protein (YAP). YAP is a transcriptional coactivator that controls the transcription of genes involved in proliferation and survival. In mouse models of Ras-driven PC, YAP is required for the formation of PanIN precursor lesions and their progression to invasive PC. In our telomerase-immortalized human pancreatic ductal cells, overexpressed YAP alone is sufficient to allow anchorage-independent growth. Our results show that in PC cells treated with Rac1 inhibitors (NSC23766, EHT-1864, AZA1, and Rac1T17N), YAP is rapidly and almost completely degraded within 24 hours. Importantly, this degradation of YAP is blocked by the preincubation of the cells with proteasome inhibitor MG132, thereby implying the involvement of a ubiquitin ligase complex. YAP contains a phosphodegron that controls its ubiqutination and subsequent degradation by the proteasome. Activation of this degron recognized by SCF-βTRCP ubiquitin ligase complexes requires its phosphorylation by the LATS1/2 kinases. To identify the molecular signals that control the degradation of YAP after Rac1 inhibition, Flag-tagged mutants of YAP were tested that were missing either a functional degron (ΔβTRCP mutant) or all of their LATS1/2 phosphorylation sites (5SA mutant). Our results show that while the functionality of the degron is required for YAP degradation after Rac1 inhibition, the phosphorylation of this degron by the LATS1/2 kinases is needed. Not only was the 5SA YAP mutant still degraded after Rac1 inhibition, but the knockdown of the LATS1/2 kinases also did not prevent the degradation of YAP. These findings suggest the existence of a novel SCF ubiquitin ligase complex that regulates YAP stability independently of the LATS1/2 kinases. The screening of a siRNA library has now been undertaken to identify the F-box protein involved. The proposed work is expected to shed light on novel mechanisms underlying the development of PC and of other Ras-driven malignancies. Citation Format: Chitra Palanivel, Bailey M. Gabler, Ying Yan, Surinder K. Batra, Michel M. Ouellette. The small GTPase Rac1 controls the stability of Yes-Associated Protein (YAP) independently of the LATS1/2 kinases [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A32.

TIMP1 down-regulation enhances gemcitabine sensitivity and reverses chemoresistance in pancreatic cancer

The therapeutic effect of gemcitabine (GEM) in pancreatic ductal adenocarcinoma (PDAC) is limited due to low drug sensitivity and high drug resistance. Tissue inhibitor of matrix metalloprotease 1 (TIMP1) is reportedly associated with GEM resistance in PDAC. However, the effect of TIMP1 down-regulation in combination with GEM treatment is unknown. We analyzed the expression of TIMP1 in human PDAC tissue using western blot, quantitative real-time polymerase chain reaction (qRT-PCR), and immunohistochemistry. TIMP1 was highly expressed in PDAC specimens. Kaplan-Meier survival analysis suggested that a higher level of TIMP1 was correlated with poorer overall survival in 103 PDAC patients. The mRNA and protein expression profiles of TIMP1 were explored in the HTERT-HPNE human pancreatic ductal epithelium cell line, five PDAC cell lines (MIA PaCa-2, PANC-1, BxPC-3, Capan2, and SW1990), and two GEM-resistant PDAC cell lines (MIA PaCa-2R and PANC-1R). Compared with HTERT-HPNE, TIMP1 was highly expressed in the PDAC cell lines. In addition, TIMP1 was upregulated in GEM-resistant PDAC cell lines compared with their parental cells. When TIMP1 was knocked-down using short hairpin RNA, GEM-induced cytotoxicity and apoptosis were increased, while colony formation was repressed in MIA PaCa-2, PANC-1, and their GEM-resistant cells. When Bax was activated by BAM7 or Bcl-2 was inhibited by venetoclax, CCK-8 assays demonstrated that GEM sensitivity was restored in GEM-resistant cells. When Bax was down-regulated by siRNA, CCK-8 assays verified that GEM sensitivity was decreased in PDAC cells. The observations that TIMP1 knockdown enhanced GEM sensitivity and reversed chemoresistance by inducing cells apoptosis indicated cooperative antitumor effects of shTIMP1 and GEM therapy on PDAC cells. The combination may be a potential strategy for PDAC therapy.

Single-cell resolution analysis of the human pancreatic ductal progenitor cell niche

We have described multipotent progenitor-like cells within the major pancreatic ducts (MPDs) of the human pancreas. They express PDX1, its surrogate surface marker P2RY1, and the bone morphogenetic protein (BMP) receptor 1A (BMPR1A)/activin-like kinase 3 (ALK3), but not carbonic anhydrase II (CAII). Here we report the single-cell RNA sequencing (scRNA-seq) of ALK3bright+-sorted ductal cells, a fraction that harbors BMP-responsive progenitor-like cells. Our analysis unveiled the existence of multiple subpopulations along two major axes, one that encompasses a gradient of ductal cell differentiation stages, and another featuring cells with transitional phenotypes toward acinar tissue. A third potential ducto-endocrine axis is revealed upon integration of the ALK3bright+ dataset with a single-cell whole-pancreas transcriptome. When transplanted into immunodeficient mice, P2RY1+/ALK3bright+ populations (enriched in PDX1+/ALK3+/CAII- cells) differentiate into all pancreatic lineages, including functional β-cells. This process is accelerated when hosts are treated systemically with an ALK3 agonist. We found PDX1+/ALK3+/CAII- progenitor-like cells in the MPDs of types 1 and 2 diabetes donors, regardless of the duration of the disease. Our findings open the door to the pharmacological activation of progenitor cells in situ.

Andrographolide promotes pancreatic duct cells differentiation into insulin-producing cells by targeting PDX-1

Regeneration of β-cells by differentiation of pancreatic progenitor cells has the potential to fundamentally solve the problems of the loss of β-cell function and mass during disease progression in both type 1 or 2 diabetes. Therefore, discovery of novel differentiation inducers to promote islet regeneration is of great significance. Pancreatic and duodenal homeobox1 (PDX-1) is a key transcription factor that promotes the development and maturation of pancreatic β-cells. To screen potential novel small molecules for enhancing differentiation of PNAC-1 cells, a human pancreatic ductal cell lines into insulin-producing cells (IPCs), we developed a high-throughput screening method through fusing the PDX-1 promoter region with a luciferase reporter gene. We screened and identified that andrographolide named C1037 stimulates PDX-1 expression in both mRNA and protein level and significantly promotes PANC-1 cells differentiation into IPCs as compared with that of control cells. The therapeutic effect of C037 in Streptozotocin induced diabetic mouse model through differentiation of pancreatic ductal cells into insulin positive islets was also observed. Our study provides a novel method to screen compounds regulating the differentiation of pancreatic progenitor cells having the potential of enhancing islet regeneration for diabetes therapy.

Branched-chain amino acids sustain pancreatic cancer growth by regulating lipid metabolism

Branched-chain amino acid (BCAA) catabolism and high levels of enzymes in the BCAA metabolic pathway have recently been shown to be associated with cancer growth and survival. However, the precise roles of BCAA metabolism in cancer growth and survival remain largely unclear. Here, we found that BCAA metabolism has an important role in human pancreatic ductal adenocarcinoma (PDAC) growth by regulating lipogenesis. Compared with nontransformed human pancreatic ductal (HPDE) cells, PDAC cells exhibited significantly elevated BCAA uptake through solute carrier transporters, which were highly upregulated in pancreatic tumor tissues compared with normal tissues. Branched-chain amino-acid transaminase 2 (BCAT2) knockdown markedly impaired PDAC cell proliferation, but not HPDE cell proliferation, without significant alterations in glutamate or reactive oxygen species levels. Furthermore, PDAC cell proliferation, but not HPDE cell proliferation, was substantially inhibited upon knockdown of branched-chain α-keto acid dehydrogenase a (BCKDHA). Interestingly, BCKDHA knockdown had no significant effect on mitochondrial metabolism; that is, neither the level of tricarboxylic acid cycle intermediates nor the oxygen consumption rate was affected. However, BCKDHA knockdown significantly inhibited fatty-acid synthesis, indicating that PDAC cells may utilize BCAAs as a carbon source for fatty-acid biosynthesis. Overall, our findings show that the BCAA metabolic pathway may provide a novel therapeutic target for pancreatic cancer.

USP21 deubiquitinase promotes pancreas cancer cell stemness via Wnt pathway activation.

The ubiquitin-specific protease (USP) family is the largest group of cysteine proteases. Cancer genomic analysis identified frequent amplification of USP21 (22%) in human pancreatic ductal adenocarcinoma (PDAC). USP21 overexpression correlates with human PDAC progression, and enforced expression of USP21 accelerates murine PDAC tumor growth and drives PanIN to PDAC progression in immortalized human pancreatic ductal cells. Conversely, depletion of USP21 impairs PDAC tumor growth. Mechanistically, USP21 deubiquitinates and stabilizes the TCF/LEF transcription factor TCF7, which promotes cancer cell stemness. Our work identifies and validates USP21 as a PDAC oncogene, providing a potential druggable target for this intractable disease.

The Histone Demethylase KDM3A, Increased in Human Pancreatic Tumors, Regulates Expression of DCLK1 and Promotes Tumorigenesis in Mice

The histone lysine demethylase 3A (KDM3A) demethylates H3K9me1 and H3K9Me2 to increase gene transcription and is upregulated in tumors, including pancreatic tumors. We investigated its activities in pancreatic cancer cell lines and its regulation of the gene encoding doublecortin calmodulin-like kinase 1 (DCLK1), a marker of cancer stem cells. We knocked down KDM3A in MiaPaCa-2 and S2-007 pancreatic cancer cell lines and overexpressed KDM3A in HPNE cells (human noncancerous pancreatic ductal cell line); we evaluated cell migration, invasion, and spheroid formation under hypoxic and normoxic conditions. Nude mice were given orthotopic injections of S2-007 cells, with or without (control) knockdown of KDM3A, and HPNE cells, with or without (control) overexpression of KDM3A; tumor growth was assessed. We analyzed pancreatic tumor tissues from mice and pancreatic cancer cell lines by immunohistochemistry and immunoblotting. We performed RNA-sequencing analysis of MiaPaCa-2 and S2-007 cells with knockdown of KDM3A and evaluated localization of DCLK1 and KDM3A by immunofluorescence. We analyzed the cancer genome atlas for levels of KDM3A and DCLK1 messenger RNA in human pancreatic ductal adenocarcinoma (PDAC) tissues and association with patient survival time. Levels of KDM3A were increased in human pancreatic tumor tissues and cell lines, compared with adjacent nontumor pancreatic tissues, such as islet and acinar cells. Knockdown of KDM3A in S2-007 cells significantly reduced colony formation, invasion, migration, and spheroid formation, compared with control cells, and slowed growth of orthotopic tumors in mice. We identified KDM3A-binding sites in the DCLK1 promoter; S2-007 cells with knockdown of KDM3A had reduced levels of DCLK1. HPNE cells that overexpressed KDM3A formed foci and spheres in culture and formed tumors and metastases in mice, whereas control HPNE cells did not. Hypoxia induced sphere formation and increased levels of KDM3A in S2-007 cells and in HPNE cells that overexpressed DCLK1, but not control HPNE cells. Levels of KDM3A and DCLK1 messenger RNA were higher in human PDAC than nontumor pancreatic tissues and correlated with shorter survival times of patients. We found human PDAC samples and pancreatic cancer cell lines to overexpress KDM3A. KDM3A increases expression of DCLK1, and levels of both proteins are increased in human PDAC samples. Knockdown of KDM3A in pancreatic cancer cell lines reduced their invasive and sphere-forming activities in culture and formation of orthotopic tumors in mice. Hypoxia increased expression of KDM3A in pancreatic cancer cells. Strategies to disrupt this pathway might be developed for treatment of pancreatic cancer.

Coxsackievirus-B4 Infection of Human Primary Pancreatic Ductal Cell Cultures Results in Impairment of Differentiation into Insulin-Producing Cells.

Coxsackievirus-B4 (CV-B4) E2 can persist in the pancreatic ductal-like cells (Panc-1 cell line), which results in an impaired differentiation of these cells into islet-like cell aggregates (ICA). In this study, primary pancreatic ductal cells obtained as a by-product of islet isolation from the pancreas of seven brain-dead adults were inoculated with CV-B4 E2, followed-up for 29 days, and the impact was investigated. Viral titers in culture supernatants were analyzed throughout the culture. Intracellular viral RNA was detected by RT-PCR. Levels of ductal cell marker CK19 mRNA and of insulin mRNA were evaluated by qRT-PCR. The concentration of c-peptide in supernatants was determined by ELISA. Ductal cells exposed to trypsin and serum-free medium formed ICA and resulted in an increased insulin secretion. Ductal cells from five brain-dead donors were severely damaged by CV-B4 E2, whereas the virus persisted in cultures of cells obtained from the other two. The ICAs whose formation was induced on day 14 post-inoculation were scarce and appeared tiny in infected cultures. Also, insulin mRNA expression and c-peptide levels were strongly reduced compared to the controls. In conclusion, CV-B4 E2 lysed human primary pancreatic ductal cells or persisted in these cells, which resulted in the impairment of differentiation into insulin-producing cells.

Abstract 1725: Mutationally activated KRAS(G12D) upregulates the expression of axonal guidance cue Semaphorin-5A in pancreatic cancer

Abstract Mutation of KRAS is commonly found in different human cancers and is generally responsible for driving tumorigenesis. Unfortunately, direct inhibitors of KRAS are not available. Thus, scientists are in search of alternative strategies that can repress the oncogenic properties of KRAS and can benefit a large number of cancer patients. Drugs targeting KRAS downstream effectors are under investigation but are facing numerous challenges in their clinical implementation. Accumulating evidence suggests that the genetic status of KRAS can modulate the role of axonal guidance cue family members in tumorigenesis. Previous reports from our laboratory have identified upregulation of one such molecule Semaphorin-5A (SEMA5A) in murine model based on KRAS gene mutation in pancreatic progenitor cells that bear a striking similarity to the human PDAC condition. We utilized PDX-cre-LSL-Kras(G12D) (KC) for studying Sema5A expression and observed no expression of Sema5A in the normal pancreas, derived from the control PDX-cre mice, however, we observed Sema5A expression in PDX-cre-LSL-Kras(G12D) mice beginning at ten weeks of age. Furthermore, we observed an increase in SEMA5A expression in the tumors of mice aged at twenty-five and fifty weeks, representing a fully developed PDAC condition. To investigate the relationship between KRAS status and SEMA5A, we assessed whether KRAS(G12D) alters the expression of SEMA5A using immortalized human pancreatic ductal cells having endogenous wild-type KRAS but an exogenous expression of KRAS(G12D) [HPNE-KRAS]. We detected higher expression of SEMA5A in the KRAS(G12D) bearing HPNE-KRAS cells in comparison with their control counterpart HPNE-Tert. Stable clones of CD18/HPAF cell line knocked-down for KRAS(G12D) demonstrated inhibition of SEMA5A expression in comparison with their respective Control CD18/HPAF cells bearing an endogenous expression of mutant KRAS. We also analyzed SEMA5A expression in the cells lines derived from the serial passage of COLO 357 cells through mice to achieve highly invasive/metastatic variants also leading to the selection of cells that express mutationally activated KRAS. L3.3 cells, with no point mutations in the KRAS gene, expressed lower SEMA5A in comparison with highly metastatic L3.6pl cells bearing a point mutation in codon 12, thereby encoding a mutationally activated KRAS(G12D). Together, our data suggest that KRAS(G12D) mutation directly upregulates the expression of axon guidance cue molecule-SEMA5A in the PDAC cells. Our findings open an avenue of molecules that can serve as secondary targets to repress KRAS activity in pancreatic cancer. Citation Format: Sugandha Saxena, Babita Tomar, Lingyun Wu, Mohammad Awaji, Satyanarayan Rachagani, Michel J. Ouellette, Surinder K. Batra, Bhavana J. Dave, Rakesh K. Singh. Mutationally activated KRAS(G12D) upregulates the expression of axonal guidance cue Semaphorin-5A in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1725.

Effect of microRNA-19b on proliferation, migration and invasion of pancreatic ductal adenocarcinoma

Objective To examine the expression of microRNA (miRNA, miR)-19b in pancreatic ductal adenocarcinoma (PDAC) cell lines, and the functional role of miR-19b in proliferation, migration and invasion of PDAC cells. Methods Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to detect the expression level of miR-19b in human pancreatic ductal epithelial cell line (HPDE), human PDAC cell lines (PANC1, MiaPaCa2 and BxPC3) and huamn PDAC metastasis cell line (COLO357) and its fast-growing variant FG. The expression of miR-19b was inhibited or overexpressed in PDAC cell lines. Transwell assay and Matrigel assay were carried out to examine the migration and invasion capabities of PDAC cells with miR-19b silencing or overexpressing. Methyl thiazol tetrazolium (MTT) and colony formation assays were performed on PDAC cell lines with miR-19b silencing or overexpression. Western blotting was used to detect the expression of proliferation markers (p21 and p27) in PDAC cell lines after miR-19b was silenced or overexpressed. Results The results of RT-qPCR showed the expression level of miR-19b in human PDAC cell lines PANC1, MiaPaCa2, BxPC3 and metastatic cell line COLO357 and FG (198.45±21.22, 221.37±18.59, 182.51±13.48, 317.64±15.20, 331.34±17.18) was significantly higher than that in normal human pancreatic ductal HPDE (98.21±14.54, P<0.05). Transwell cell migration assay and Matrigel cell invasion assay showed that the migration and invasion ability of FG cells after miR-19b silencing [(22.54±2.87)%, (28.87±5.62)%] was significantly decreased as compared with the control group [(99.65±3.07)%, (98.73±4.38)%, P<0.01]. In contrast, the migration and invasion ability of BxPC3 cells overexpressing miR-19b [(317.53±10.21)%, (286.77±12.83)%] was significantly higher than that of the control group [(97.89±5.31)%, (98.25±7.57)%, P<0.01]. The colony formation and MTT assays showed that the proliferation ability of PDAC cells after miR-19b was silenced was significantly decreased as compared with the control group (P<0.01). The result of Western blotting showed that the expression of proliferation markers (p21 and p27) in PDAC was increased by (3.25±0.27) and (5.38±0.33) times respectively as compared with the control group after miR-19b silencing (P<0.01). When miR-19b was overexpressed, the expression of p21 and p27 decreased by (2.68±0.15) and (3.27±0.27) times respectively as compared with the control group (P<0.01). Conclusion MiR-19b could promote the migration and invasion of PDAC cells and increase the proliferation capability. Key words: Pancreatic carcinoma; MicroRNA-19b; Metastsis; Proliferation

In vivo imaging of insulin-secreting human pancreatic ductal cells using MRI reporter gene technique: A feasibility study.

The purpose of this study was to investigate the feasibility of in vivo imaging of human pancreatic ductal cells by OATP1B3 reporter gene under MRI. A human cell line (PANC-1) derived from the pancreatic ductal epithelium was used in this study. After transduction of OATP1B3, the cellular physiological functions and the ability of intracellular uptake of the MRI contrast medium (Gd-EOB-DTPA) were examined. Induced differentiation of the PANC-1 cells into hormone-secreting cells were performed to simulate pancreatic β-like cells. The hormone-secreting cells were implanted into rats and in vivo MRI was evaluated. The mRNA and proteins of OATP1B3 were highly expressed. No significant change of cellular physiological functions was found after the expression. After induced differentiation, the hormone secretion capacities of the OATP1B3-expressing PANC-1 cells were confirmed. Intra-cellular uptake of Gd-EOB-DTPA was determined in vitro by inductively coupled plasma mass spectrometry and MRI. In vivo MRI of the OATP1B3-expressing xenograft revealed an increased signal intensity after contrast enhancement. OATP1B3 can be used as a safe and feasible in vivo MRI gene reporter for human pancreatic ductal cells.

Studies of cell surface and soluble HLA class I expression in pancreatic beta cells exposed to interferons or Poly I:C

HLA class I (HLA-I) molecules play a crucial role in cell-mediated immunity by presenting peptide antigens to cytotoxic CD8+T cells. A pathological hallmark of Type 1 diabetes (T1D) is the hyperexpression of HLA-I in pancreatic islets that contain residual insulin producing beta cells. The expression of HLA-I can be induced following exposure to interferons (IFN). Previous studies have implicated enteroviruses (EV) as major players in triggering an autoimmune response against beta cells and it seems likely that the hyperexpression of HLA-I contributes to the recognition and targeting of beta cells by CD8+T cells. Whilst HLA-I molecules are expressed on the surface of nucleated cells, there is increasing evidence that HLA-I can also be found in a soluble form in the plasma, including in patients with viral infections. Intriguingly, soluble HLA-I (sHLA-I) levels are significantly elevated in the serum of patients with autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis and T1D, yet the function of sHLA-I is still unclear. In this project, we are investigating the impact of interferons and poly I:C, a viral dsRNA mimetic, on the expression of surface and soluble HLA-I in the human pancreatic beta cell line EndoC-βH1, the human pancreatic ductal cell line PANC1 and in HeLa cells. We show that surface HLA-I is upregulated in response to these stimuli in each of the cell lines, with greatest magnitude in EndoC-βH1. We also show for the first time that release of sHLA-I is significantly increased in response to IFNγ in EndoC-βH1 cell line.

Impact of gold nanoparticles shape on their cytotoxicity against human osteoblast and osteosarcoma in in vitro model. Evaluation of the safety of use and anti-cancer potential

Due to development of nanotechnology and gold nanoparticles (AuNPs) increasing use in different areas of medicine, especially in oncology, better understanding of their potential cytotoxicity is necessary to protect patients safety. Shape and size of AuNPs is an important modulator of their cytotoxicity. Therefore, we investigated the cytotoxicity of AuNPs rods (≈39 nm length, 18 nm width), AuNPs stars (≈ 215 nm) and AuNPs spheres (≈ 6.3 nm) against human fetal osteoblast (hFOB 1.19), osteosarcoma (143B, MG63) and pancreatic duct cell (hTERT-HPNE) lines by MTT and neutral-red uptake assay. Moreover, influence of AuNPs on level of proapoptotic protein (Bax) and anti-apoptotic protein (Bcl-2) was measured by western blot. Cellular uptake of nanoparticles and ultrastructure changes were examined by transmission electron microscopy (TEM). In the present study we have proven that AuNPs stars are the most cytotoxic against human cells. We observed that cancer cells are more susceptible to AuNPs cytotoxic effect. Furthermore, AuNPs rods and AuNPs stars caused increased expression of Bax and decreased expression of Bcl-2 protein in osteosarcoma cells. We found that AuNPs penetrated through the cell membrane and caused ultrastructural changes. Our results clearly demonstrated that the cytotoxicity of AuNPs was shape-dependent. AuNPs stars with the highest anti-cancer potential were also the most cytotoxic type of tested NPs, whereas AuNPs spheres which appears to be the safest one had small anti-cancer potential.

Spheroid Culture of Human Pancreatic Ductal Cells to Reconstitute Development of Pancreatic Intraepithelial Neoplasia.

Pancreatic ductal adenocarcinoma (PDA) presents poor 5-year survival rate, mainly attributable to late diagnosis due to its asymptomatic nature. Therefore, building human cell-based systems that reconstitute hallmark features of the PDA precursors, pancreatic intraepithelial neoplasia (PanINs), will accelerate development of new strategies for early diagnostics and intervention. We previously demonstrated that systematic introduction of genetic modification (KRAS, CDKN2A, SMAD4, and TP53) leads to immortalization of primary human pancreatic cells and, upon orthotopic transplantation, their development to human PanIN-like lesions. Here, we describe detailed methods for fluorescence-activated cell sorting, lentiviral transduction, and three-dimensional spheroid culture of primary adult human pancreatic ductal cells, as well as a method for clonal selection of human pancreatic ductal spheres.

Lineage Tracing of Primary Human Pancreatic Acinar and Ductal Cells for Studying Acinar-to-Ductal Metaplasia.

Acinar-to-ductal metaplasia may play important roles in the development of various pancreatic diseases. Here, we describe a method to induce ADM in primary human cells in 3D culture. We developed a flow cytometry lineage tracing strategy to identify and sort viable acinar, ductal, and acinar-derived ductal-like cells for further molecular and functional analysis.

Porphyrins to restrict progression of pancreatic cancer by stabilizing KRAS G-quadruplex: In silico, in vitro and in vivo validation of anticancer strategy

KRAS, a frequently mutated G-quadruplex forming proto-oncogene is responsible for almost every type of cancer which can form a parallel G-quadruplex structure in the promoter region. G-quadruplex structure is one of the most important drug targets for modern cancer therapy for their unique structure and specificity. Here, we have screened several synthetic porphyrin-based compounds as potential KRAS G-quadruplex stabilizing ligands, using molecular modeling and docking studies. Two novel porphyrins: Porphyrin-1(Cobalt containing) and Porphyrin-2 (Palladium containing) evidenced high affinity towards KRAS-promoter/G-quadruplex. As KRAS mutation is prevalent in pancreatic cancer, the efficacy of these ligands against human pancreatic ductal carcinoma cell line PANC-1 and MiaPaCa2 were examined. Both the Porphyrins exhibited significant cytotoxicity and block metastasis by inhibiting Epithelial to messenchymal transition. In vivo studies confirmed both porphyrin compounds to be effective against EAC tumors along with significantly low toxicity against normal Swiss albino mice. The expression of KRAS gene in porphyrin-treated PANC-1, MiaPaCa2 and tumor-derived EAC cells were drastically reduced at both protein and RNA levels. Thus interaction of porphyrin-based ligands with G-quadruplex DNA at the promoter region of KRAS, might be utilized as a target for anticancer therapeutic strategy.

Role of microRNA-19b in proliferation and metastasis of pancreatic ductal adenocarcinoma

Objective To examine the expression of miR-19b in pancreatic ductal adenocarcinoma (PDAC) cell lines, and invetigate the functional role of miR-19b in proliferation, migration and invasion of PDAC cells. Methods Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to detect the expression level of miR-19b in human pancreatic ductal epithelial cell line (HPDE), human PDAC cell lines (PANC1, MiaPaCa2 and BxPC3) and huamn PDAC metastasis cell line COLO357 and its fast-growing variant FG. PDAC cell lines stably over-expressing of low expressing miR-19b were constructed. Transwell assay and Matrigel assay were carried out to examine the migration and invasion capabities of PDAC cells with miR-19b down-regulated or up-regulated. Methyl thiazol tetrazolium (MTT) and colony formation assay were performed on PDAC cell lines with miR-19b silenced or overexpressed. Results The results of RT-qPCR showed the expression level of miR-19b in human PDAC cell lines PANC1, MiaPaCa2, BxPC3 and metastatic cell line COLO357 and FG (198.45±21.22, 221.37±18.59, 182.51±13.48, 317.64±15.20, 331.34±17.18) was significantly higher than that in HPDE (98.21±14.54), (P=0.003, 0.005, 0.021, 0.001, 0.001) and the difference was statistically significant. Transwell cell migration assay and Matrigel cell invasion assay revealed that the migration and invasion ability of FG cells after miR-19b silencing [(22.54±2.87)%, (28.87±5.62)%] was significantly decreased as compared with the control group [(99.65±3.07)%, (98.73±4.38)%], (P=0.005, 0.002) and the difference was statistically significant. In contrast, the migration and invasion ability of BxPC3 cells overexpressing miR-19b [(317.53±10.21)%, (286.77±12.83)%] was significantly higher than that in the control group [(97.89±5.31)%, (98.25±7.57)%] (P=0.003, 0.007) and the difference was statistically significant. The colony formation and MTT assay showed that after miR-19b silencing, the expression of p21 and p27 in PDAC cells was significantly reduced as compared with the control group (P=0.003, 0.005) and the difference was statistically significant. The results of Western blotting indicated that the silencing of miR-19b resulted in the up-regulation of p21 and p27 in PDAC cells by (3.25±0.27, 5.38±0.33) as compared with the control group (P=0.003, 0.001) and the difference was statistically significant. The overexpression of miR-19b led to the down-regulation of p21 and p27 by (2.68±0.15, 3.27±0.27) as compared with the control group (P=0.006, 0.005) and the difference was statistically significant. Conclusion MiR-19b could promote the migration and invasion of PDAC cells and increase the proliferation capability. Key words: MicroRNA-19b; Pancreatic ductal adenocarcinoma; Metastsis; Proliferation