Human Pancreatic Ductal Epithelial Cells Research Articles

The expression of long non-coding RNA-LINC01410 in pancreatic cancer and its effect on proliferation and migration of pancreatic cancer cells

Objective: To explore the expression of long non-coding RNA-LINC01410 in the pancreatic cancer tissues, corresponding paracancerous tissues, human pancreatic cancer cell lines and normal pancreatic ductal epithelial cell line, and analyze the effect of LINC01410 on pancreatic cancer cell proliferation and migration. Methods: Real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the expression level of LINC01410 in 16 cases of pancreatic cancer tissue and its adjacent tissues. RT-qPCR was performed to analyze LINC01410 expression in the pancreatic cancer cell lines AsPC-1, CAPAN-1, SW1990, BxPC-3 and CFPAC-1, and human normal pancreatic ductal epithelial cell line HPDE6-C7. Transfection of interference plasmid (shRNA) in the pancreatic cancer cell line with the highest expression level of LINC01410 were used to knock-down the expression of LINC01410. CCK-8 assay, colony formation assay, and transwell chamber assay were performed to detect the proliferation and migration of pancreatic cancer cells. The complementary paired miRNAs and downstream genes of LINC01410 were predicted by bioinformatics. The expression of miRNA and downstream genes was detected by RT-qPCR, and the protein expression of downstream genes was determined by Western blot. Results: The expression of LINC01410 in the pancreatic cancer tissues was significantly higher than that in the adjacent tissues [(3.46±0.32) vs (0.65±0.08), P<0.01]. The expression levels of LINC01410 in the pancreatic cancer cell lines were significantly higher than that in the normal human pancreatic ductal epithelial cells (P<0.05). The expression of LINC01410 was highest in BxPC-3 cells (P<0.01). After knock-down of the LINC01410 expression in the pancreatic cancer cell line BxPC-3, the cell proliferation was significantly inhibited (P<0.05), and the cell migration ability was decreased (P<0.05). LINC01410 complementarily paired with miR-497-5p, and miR-497-5p complementarily paired with IFITM3. After inhibiting the expression of LINC01410, the expression of miR-497-5p was increased [(1.04±0.17) vs (5.79±0.43), P<0.01], the mRNA expression of IFITM3 was decreased [(0.39±0.05) vs (1.00±0.03), P<0.01], and the protein expression of IFITM3, CDK6, Cyclin D2, PCNA, Vimentin, and N-cadherin was decreased. Conclusions: The expression of LINC01410 was increased in pancreatic cancer tissues and cell lines. Down-regulation of LINC01410 expression inhibits the proliferation and migration of pancreatic cancer BxPC-3 cells, and its mechanism may be closely related to regulating the miR-497-5p and IFITM3 gene expression.

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

Pimavanserin tartrate: A potential drug for pancreatic cancer therapy in future

Despite major advances in cancer treatment, pancreatic cancer is still incurable and the treatment outcomes are limited. In the current study, we evaluated the anti‐cancer effects of pimavanserin tartrate (PVT), a drug used for the treatment of Parkinson's disease psychosis. Our observations indicated that, PVT significantly suppressed the proliferation of pancreatic cancer cells by inducing apoptosis without exerting any cytotoxic effects in normal human pancreatic ductal epithelial (HPDE‐6) cells. The colony forming ability of pancreatic cancer cells was significantly inhibited with PVT treatment. Anti‐proliferative and apoptosis inducing effects of PVT were mediated by the inhibition of pAkt (Ser473), Akt, Gli1, Oct‐4, SOX‐2, NANOG and c‐Myc. Akt and Oct‐4 inhibition by PVT treatment was further validated by immunofluorescence analysis. Apoptotic effects of PVT was confirmed by increase in cleavage of caspase‐3 and PARP. Moreover, PVT inhibited the formation of tumorspheres in PANC1 pancreatic cancer cells. Pharmacologically inhibiting or genetically knocking out Akt or Gli1 enhanced the growth suppressive effects of PVT in pancreatic cancer cells. Subsequently, we evaluated the effects of PVT in gemcitabine‐resistant cells. Our results demonstrated that, PVT reduced the survival of MIAPaCa2 gemcitabine‐resistant cells in a concentration and time‐dependent manner. Further mechanistic analysis indicated that, PVT suppressed the phosphorylation of Akt at Ser 473 and inhibited the expression of Gli1, Oct‐4 and c‐Myc in MIAPaCa2 gemcitabine‐resistant cells. Moreover, PVT increased the cleavage of caspase‐3 and PARP as an indicator of apoptosis. Oral administration of PVT suppressed BxPC3 tumor xenografts by 50% in athymic nude mice. In another in vivo experiment, PVT treatment inhibited the growth of orthotopically implanted PANC1 tumors by 75%. Chronic administration of PVT did not exhibit any general signs of toxicity or behavioral side effects in mice. Taken together, our results indicate that pancreatic tumor growth suppression by PVT is orchestrated by inhibition of Akt/Gli1 signaling. Since PVT is already available in the clinic with an established safety profile, our results will accelerate its clinical development for the treatment of patients with pancreatic cancer.Support or Funding InformationSupported in part by R01 grant CA129038, awarded to (S.K.S.) by the National Cancer Institute, Syngenta fellowship grant awarded by Syngenta pharmaceuticals.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Knockdown of ISOC1 suppresses cell proliferation in pancreatic cancer in vitro.

Pancreatic cancer is a deadly disease that is frequently associated with mortality at the time of diagnosis due to rapid metastasis, which makes it unsuitable for operative surgery, and resistant to chemotherapy and radiation therapy. Isochorismatase domain-containing protein 1 (ISOC1) has putative isochorismatase activity, and is positively regulated by estrogen in human breast cancer. However, its role in pancreatic cancer has yet to be fully elucidated. Analysis from datasets downloaded from The Cancer Genome Atlas and Genotype-Tissue Expression databases indicated that the ISOC1 mRNA expression level was increased in pancreatic cancer tissues, compared with normal pancreatic tissues. In the present study, it was determined that the human pancreatic cancer cell lines SW 1990, PANC-1 and AsPC-1 had increased expression levels of ISOC1 mRNA, compared with human pancreatic ductal epithelial cells. Additionally, two of the pancreatic cancer cell lines, SW 1990 and PANC-1, transfected with lentivirus-delivered short hairpin RNA, to knockdown the expression of ISOC1, were established. Cell counting and MTT assays indicated that knockdown of ISOC1 decreased the ability of cell growth and proliferation in pancreatic cancer cells. Furthermore, Annexin V staining and caspase-3/7 activity assays demonstrated that inhibition of ISOC1 promoted cell apoptosis via elevation of the expression of caspase-3/7. Furthermore, inhibition of ISOC1 impaired the cell migration and invasive capability of the cells. In conclusion, ISOC1 exerts a role in pancreatic cancer cell growth and apoptosis, and may have a role in pancreatic cancer tumorigenesis.

Knockdown of KDM1B inhibits cell proliferation and induces apoptosis of pancreatic cancer cells

Pancreatic cancer (PC) is one of the common malignant tumors in digestive tract with a high fatality rate. The oncogenic role of lysine-specific demethylase1 (LSD1/KDM1 A) has been well recognized in PC. While, the role of its homolog LSD2 (KDM1B) in regulating PC progression is poorly understood. In this study, we attempted to evaluate the functional role of KDM1B in PC cells. The expression of KDM1B was detected by immunohistochemistry and immunoblotting in PC tissues and cells. Lentivirus-mediated shRNA was applied to silence KDM1B in PANC-1 and SW1990 cells. Cell proliferation was measured by MTT and Celigo assay. Cell apoptosis was determined by both Caspase-Glo®3/7 assay and Flow cytometry. Intracellular signaling molecules were detected using a PathScan intracellular signaling array kit. In this study, we found KDM1B was highly expressed in PC tissues compared to paracancerous tissues. Moreover, elevated expression of KDM1B was detected in PC cell lines (BxPC-3, CFPAC-1, PANC-1 and SW1990) as compared with a normal human pancreatic duct epithelial cell line (HPDE6-C7). Further investigations revealed that KDM1B knockdown significantly inhibited PC cell proliferation. Furthermore, the apoptosis of PANC-1 and SW1990 cells was significantly increased after KDM1B knockdown. Notably, the activations of p-ERK1/2, p-Smad2, p-p53, cleaved PARP, cleaved Caspase-3, cleaved Caspase-7, p-eIF2a and Survivin were promoted by KDM1B knockdown, while IkBa was suppressed. Taken together, our findings provided new insights into the critical and multifaceted roles of KDM1B in the regulation of cell proliferation and apoptosis, and offered a potentially novel target in preventing the progression of PC.

Inhibition of pancreatic cancer stem cell characteristics by α-Mangostin: Molecular mechanisms involving Sonic hedgehog and Nanog.

The current investigation was intended to elucidate the molecular mechanism of α‐Mangostin in the regulation of pancreatic cancer stem cell (CSC) characteristics. Here, we demonstrate that α‐Mangostin inhibited cell proliferation in pancreatic CSCs and cancer cell lines while it showed no effect on human pancreatic normal ductal epithelial cells. Also, α‐Mangostin inhibited colony formation and induced apoptosis in these cells. Further, α‐Mangostin inhibited the self‐renewal capacity of CSCs isolated from human primary tumours and KrasG12D mice. Furthermore, α‐Mangostin inhibited the invasive and metastatic ability of pancreatic CSCs by suppressing the epithelial‐to‐mesenchymal transition (EMT) via up‐regulation of E‐cadherin and down‐regulation of mesenchymal phenotype by inhibiting N‐cadherin, Snail and Slug expression. Interestingly, the pluripotency maintaining factors and CSC markers were inhibited by α‐Mangostin thus suggesting that α‐Mangostin can target CSCs to inhibit pancreatic cancer effectively. Gli signalling plays a crucial role in the self‐renewal and pluripotency of CSCs. α‐Mangostin inhibited the Gli transcription and the expression of Gli target genes (Nanog, Oct4, c‐Myc, Sox‐2 and KLF4) in CSCs. Using ChIP assay, we demonstrated that Nanog could directly bind to promoters of Cdk2, Cdk6, FGF4, c‐Myc and α‐Mangostin inhibited Nanog binding to these promoters. Conversely, the inhibitory effects of the α‐Mangostin on CSC proliferation and Gli or Nanog transcription and their targets were abrogated by either enforced activation of sonic hedgehog (Shh) or by the overexpression of Nanog. Taken together, our studies suggest that α‐Mangostin may act as Gli inhibitor and establishes the pre‐clinical significance of α‐Mangostin for the prevention and treatment of pancreatic cancer.

Triacetyl resveratrol upregulates miRNA‑200 and suppresses the Shh pathway in pancreatic cancer: A potential therapeutic agent.

Trans‑3,4',5‑trihydroxystilbene (resveratrol) is a naturally occurring polyphenolic phytoalexin with marked anticancer activities, and is mainly found in grapes, berries and peanuts. However, due to a low bioavailability, it has not progressed to clinical practice for cancer treatment. Therefore, the aims of the present study were to examine the anticancer activities of the resveratrol derivative, triacetyl resveratrol(TCRV), in pancreatic cancer cells. Apoptosis was measured by fluorescence‑activated cell sorting and terminal deoxynucleotidyl transferase(TdT)‑mediated dUTP nick‑end labeling assays. Gene expression was measured by reverse transcription‑quantitative polymerase chain reaction. TCRV inhibited colony formation and induced apoptosis through caspase‑3 activation in human pancreatic cancer AsPC‑1 and PANC‑1 cells, whereas it exerted no effect on human pancreatic normal ductal epithelial cells (HPNE). TCRV inhibited epithelial‑mesenchymal transition (EMT) by upregulating the expression of E‑cadherin and suppressing the expression of N‑cadherin and the transcription factors, Snail, Slug and Zeb1. TCRV inhibited Zeb1 3'UTR‑luciferase activity through the upregulation of microRNA (miR)‑200 family members. The inhibitory effects of TCRV on pancreatic cancer cell migration and invasion were counteracted by anti‑miR‑200 family members. The inhibitory effects of TCRV on EMT and the induction of apoptosis were exerted through the suppression of the sonic hedgehog (Shh) pathway, and through the modulation of cyclinD1 and Bcl‑2 expression. The hyperactivation of the Shh pathway by either Shh protein or Gli1 overexpression abrogated the biological effects of TCRV. Taken together, the results of this study demonstrate that TCRV inhibits pancreatic cancer growth and EMT by targeting the Shh pathway and its downstream signaling mediators. TCRV inhibited EMT through the upregulation of miR‑200 family members. Since TCRV effectively inhibited the growth of human pancreatic cancer cells by modulating the Shh pathway, without affecting the growth of HPNE cells, our findings suggest the possible use of TCRV as a promising candidate for the treatment and/or prevention of pancreatic cancer.

The synergic inhibitory effects of dark tea (Camellia sinensis) extract and p38 inhibition on the growth of pancreatic cancer cells.

Background: Dark tea is one of the most popular types of Chinese tea, which has been reported to exhibit anti-obesity, anti-oxidation and antitumor activities in according human cell lines. In terms of tumorigenesis, the systemic study of the physiological effect of specific fraction of dark tea and the relevant molecular mechanism warrant more attention.Methods: Dark tea was firstly isolated through solvent extraction method. Dissolved ethyl acetate extract was further fractioned by elution with various concentration of ethyl alcohol. The cytotoxicity effect of dark tea on cell proliferation was evaluated by CCK8 assay in HPDE human normal pancreatic duct epithelial cells, SW1990 and PANC-1 human pancreatic cancer cells, and SW1116 human colorectal cancer cells. Immunoblotting and flow cytometry analysis were utilized to examine the status of protein and reactive oxygen species respectively. Gene expression profile was analyzed by cDNA microarray and real-time PCR. The plasmid for ID1 expression was stably transfected into SW1990 cells for relevant functional analysis. The effect of dark tea extract on tumorigenesis in vivo was studied in xenograft tumor model.Results: Water eluate fraction of the ethyl acetate extract from dark tea inhibited the growth of SW1990, PANC-1 and SW1116 cells more efficiently compared with that in HPDE cells. Meanwhile, p38 activity was increased and AKT activity was dropped in cancer cells with dark tea extract treatment. Further functional analyses indicated that water eluate fraction and p38 inhibitor treatment exerted a synergic inhibitory effect on cancer cells growth, which was related to their suppressive effect on expression level of ID1 (inhibitor of differentiation protein 1), which was highly expressed in cancer cells. The analysis utilizing xenograft tumor model further indicated water eluate fraction exhibited a significantly inhibitory effect on tumorigenesis.Conclusion: Based on the sequential extraction procedure, our results reveal the inhibitory effect of water eluate fraction of the ethyl acetate extract from dark tea and its synergistic effect with p38 inhibition on the growth of pancreatic cancer cells, in which ID1 is identified as a downstream effector. This sheds insights into the physiological relevance of specific fraction of dark tea to tumorigenesis in pancreatic cancer.

Hot water extract of Agaricus blazei Murrill specifically inhibits growth and induces apoptosis in human pancreatic cancer cells

BackgroundPancreatic cancer is one of the most aggressive human malignancies. The development of a novel drug to treat pancreatic cancer is imperative, and it is thought that complementary and alternative medicine (CAM) could yield such a candidate. Agaricus blazei Murrill is a CAM that has been tested as an anticancer drug, but its efficacy against pancreatic cancer is poorly understood. To study the potential of A. blazei in the treatment of pancreatic cancer, we examined the effects of its hot water extract on the proliferation and global gene expression profile of human pancreatic cancer cells.MethodsThree distinct human pancreatic cancer cell lines, MIAPaCa-2, PCI-35, and PK-8, and the immortalized human pancreatic duct-epithelial cell line, HPDE, were employed. The cells were incubated with the appropriate growth medium supplemented with the hot water extract of A. blazei at final concentrations of 0.005, 0.015%, or 0.045%, and cellular proliferation was assessed for five consecutive days using an MTT assay. Apoptosis was examined by using flow cytometry and the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Caspase-dependent apoptosis was assayed using immunoblotting. Global gene expression profiles were examined using a whole human genome 44 K microarray, and the microarray results were validated by using real-time reverse transcription PCR.ResultsThe hot water extract of A. blazei significantly inhibited the proliferation of cultured pancreatic cancer cells through the induction of G0/G1 cell cycle arrest and caspase-dependent apoptosis; the effect was the smallest in HPDE cells. Furthermore, significant alterations in the global gene expression profiles of pancreatic cancer cells occurred following treatment with the hot water extract of A. blazei. Genes associated with kinetochore function, spindle formation, and centromere maintenance were particularly affected, as well as cyclins and cyclin-dependent kinases that are essential for cell cycle progression. In addition, proapoptotic genes were upregulated.ConclusionsThe hot water extract of A. blazei may be useful for the treatment of pancreatic cancer and is a potential candidate for the isolation of novel, active compounds specific for mitotic spindle dysfunction.

SWI/SNF component ARID1A restrains pancreatic neoplasia formation

ObjectiveARID1A is commonly mutated in pancreatic ductal adenocarcinoma (PDAC), but the functional effects of ARID1A mutations in the pancreas are unclear. Understanding the molecular mechanisms that drive PDAC formation may...

A drug‐repositioning screen for primary pancreatic ductal adenocarcinoma cells identifies 6‐thioguanine as an effective therapeutic agent for TPMT‐low cancer cells

Pancreatic cancer is one of the most difficult cancers to cure due to the lack of early diagnostic tools and effective therapeutic agents. In this study, we aimed to isolate new bioactive compounds that effectively kill pancreatic ductal adenocarcinoma (PDAC) cells, but not untransformed, human pancreatic ductal epithelial (HPDE) cells. To this end, we established four primary PDAC cell lines and screened 4141 compounds from four bioactive‐compound libraries. Initial screening yielded 113 primary hit compounds that caused over a 50% viability reduction in all tested PDAC cells. Subsequent triplicate, dose‐dependent analysis revealed three compounds with a tumor cell‐specific cytotoxic effect. We found that these three compounds fall into a single category of thiopurine biogenesis. Among them, 6‐thioguanine (6‐TG) showed an IC50 of 0.39–1.13 μm toward PDAC cells but had no effect on HPDE cells. We propose that this cancer selectivity is due to differences in thiopurine methyltransferase (TPMT) expression between normal and cancer cells. This enzyme is responsible for methylation of thiopurine, which reduces its cytotoxicity. We found that TPMT levels were lower in all four PDAC cell lines than in HPDE or Panc1 cells, and that knockdown of TPMT in HPDE or Panc1 cells sensitized them to 6‐TG. Lastly, we used a patient‐derived xenograft model to confirm that 6‐TG has a significant antitumor effect in combination with gemcitabine. Overall, our study presents 6‐TG as a strong candidate for use as a therapeutic agent against PDAC with low levels of TPMT.

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

Abstract 3068: Chronic alcohol exposure induces malignant transformation of pancreatic ductal epithelial cells and generates stem cell characteristics

Abstract Findings from epidemiologic studies and research with experimental animal models provide insights into alcohol toxicity-related disease pathogeneses. Epidemiologic data indicate that heavy drinking is associated with high risk of pancreatic cancer. However the molecular mechanisms by which chronic ethanol exposure changes pathophysiology of human pancreatic ductal epithelial (HPNE) cells is not well understood. Cancer stem cells (CSCs) play significant role in cellular transformation, and their emergence is responsible for cancer initiation, progression and metastasis. SATB2 is required for normal mammalian development; however, it is not expressed in normal adult cells. Inappropriate activation of SATB2 gene may cause malignant cellular transformation. SATB2 regulates transcription of pluripotency maintaining factors which are required for sustaining the stem cells capacity for self-renewal. The main goal of the paper is to examine the molecular mechanisms by which chronic alcohol consumption induces cellular transformation of HPNE cells. To investigate the role of SATB2 at an early step of cell transformation, we utilized HPNE cells to generate progenitor cells either by chronic ethanol exposure or overexpression with SATB2. Our data demonstrate that chronic exposure of HPNE cells to ethanol induces cellular transformation by upregulating SATB2. Furthermore, during ethanol-induced cell transformation, cells gain the phenotype of progenitor cells / CSCs and express pluripotency maintaining factors and epithelial-mesenchymal transition (EMT) markers. Suppression of SATB2 expression in ethanol-transformed HPNE cells inhibits stemness and EMT. SATB2 is highly expressed in pancreatic cancer tissues derived from human and KrasG12D mice. In conclusion, chronic ethanol exposure may contribute in pancreatic carcinogenesis by inducing the expression of SATB2 gene. Citation Format: We Yu, Yiming Ma, Sharmila Shankar, Rakesh K. Srivastava. Chronic alcohol exposure induces malignant transformation of pancreatic ductal epithelial cells and generates stem cell characteristics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3068.

Abstract 3501: Adipocytes sustain pancreatic cancer progression through a non-canonical WNT paracrine network inducing ROR2 nuclear shuttling

Abstract Background: Solid epidemiological evidences connect obesity with incidence, stage, and survival in pancreatic cancer. However, the underlying mechanistic basis linking adipocytes to pancreatic cancer progression remain largely elusive. We hypothesized that factors secreted by adipocytes could be responsible for epithelial-to-mesenchymal transition (EMT) induction and, in turn, a more aggressive phenotype in models of pancreatic preneoplastic lesions. Methods: We studied the role of factors secreted by two adipogenic model systems from primary human Bone Marrow Stromal Cells (hBMSC) in an in vitro experimental cell transformation model system of human pancreatic ductal epithelial (HPDE) cell stably expressing activated KRAS (HPDE/KRAS). Results: We measured a significant induction of EMT and aggressiveness in HPDE and HPDE/KRAS cell lines when cultured with medium conditioned by fully differentiated adipocytes (ADIPOCM) if compared with the same cells cultured with medium conditioned by hBMSC (hBMSCCM) from two different healthy donors. Several genes coding for soluble modulators of the non-canonical WNT signaling pathway, including FRZB, SFRP2, RSPO1, WNT5A and 5B were significantly overexpressed in fully differentiated adipocytes than in their respective in hBMSC. ADIPOCM induced the overexpression and the nuclear translocation of the Frizzled family member Receptor tyrosine kinase-like orphan receptor (Ror) 2 in HPDE and HPDE/KRAS cells. Vantictumab, an anti-Frizzled monoclonal antibody, reduced ROR2 nuclear translocation and in turn the EMT and aggressiveness in HPDE and HPDE/KRAS cells. Conclusions: We demonstrated that adipocytes could induce EMT and aggressiveness in models of pancreatic preneoplastic lesions by orchestrating a complex paracrine signaling of soluble modulators of the non-canonical WNT signaling pathway that determine, in turn, the activation and nuclear translocation of ROR2. This signaling pathway could represent a novel target for pancreatic cancer chemoprevention. Most importantly, these factors could serve as novel biomarkers to select a risk population among obese subjects for screening and, thus, early diagnosis of pancreatic cancer. Citation Format: Carmine Carbone, Geny Piro, Raffaela Santoro, Francesca Simionato, Valeria Merz, Camilla Zecchetto, Aldo Scarpa, Giampaolo Tortora, Davide Melisi. Adipocytes sustain pancreatic cancer progression through a non-canonical WNT paracrine network inducing ROR2 nuclear shuttling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3501.

Abstract LB-047: Novel anti-psychotic drug for pancreatic cancer treatment

Abstract Pancreatic cancer is one of the most deadly malignancy with a five-year survival rate of 7%. Despite advances in cancer treatment, this disease is still incurable and the treatment outcomes are limited. Few studies have shown that neuroleptic drugs exert better anti-proliferative effects in pancreatic cancer cells when compared to conventional chemotherapeutic agents gemcitabine and oxaliplatin. In the current study, we evaluated the anti-cancer effects of pimavanserin, an anti-psychotic drug used for the treatment of Parkinson's disease psychosis. Pimavanserin significantly suppressed the proliferation of AsPC1, BxPC3, PANC1, MIAPaCa2 and PO2 pancreatic cancer cells with the IC50 between 3 - 9µM at 24, 48 and 72 hours. Pimavanserin treatment induced 2-5 fold apoptosis in pancreatic cancer cells. Notably, no significant cell death was observed in normal human pancreatic ductal epithelial (HPDE-6) cells by pimavanserin treatment. Furthermore, pimavanserin treatment inhibits the expression of key oncogenic proteins and cancer stem cell markers such as pAkt(S473), pSTAT3(Y705), β-catenin, GLI-1, GLI-2, c-Myc, Oct-4, SOX-2, NANOG and CD44. We further evaluated the efficacy of pimavanserin in two different in vivo tumor models. Oral administration of 10mg/kg pimavanserin suppressed 50% of sub-cutaneously implanted BxPC3 tumors in athymic nude mice. In another setting, 10mg/kg pimavanserin by oral gavage inhibited the growth of orthotopically implanted PANC1 pancreatic tumors by 75%. Moreover, chronic administration of pimavanserin did not show any general signs of toxicity and no apparent changes were observed in the behavioral activity of mice. Further mechanistic studies are in progress. Since pimavanserin is already available in the clinic with established safety profile, our results will accelerate its clinical development for treatment of patients with pancreatic cancer. Our study is the first to demonstrate the anti-cancer effects of pimavanserin. Citation Format: Sharavan Ramachandran, Sanjay K. Srivastava. Novel anti-psychotic drug for pancreatic cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-047.

A repetitive mutation and selection system for bacterial evolution to increase the specific affinity to pancreatic cancer cells.

It is difficult to target and kill cancer cells. One possible approach is to mutate bacteria to enhance their binding to cancer cells. In the present study, Gram-negative Escherichia coli and Gram-positive Bacillus subtilis were randomly mutated, and then were positively and negatively selected for binding cancer vs normal cells. With repetitive mutation and selection both bacteria successfully evolved to increase affinity to the pancreatic cancer cell line (Mia PaCa-2) but not normal cells (HPDE: immortalized human pancreatic ductal epithelial cells). The mutant E. coli and B. subtilis strains bound to Mia PaCa-2 cells about 10 and 25 times more than to HPDE cells. The selected E. coli strain had mutations in biofilm-related genes and the regulatory region for a type I pilus gene. Consistent with type I pili involvement, mannose could inhibit the binding to cells. The results suggest that weak but specific binding is involved in the initial step of adhesion. To test their ability to kill Mia PaCa-2 cells, hemolysin was expressed in the mutant strain. The hemolysin released from the mutant strain was active and could kill Mia PaCa-2 cells. In the case of B. subtilis, the initial binding to the cells was a weak interaction of the leading pole of the motile bacteria. The frequency of this interaction to Mia PaCa-2 cells dramatically increased in the evolved mutant strain. This mutant strain could also specifically invade beneath Mia PaCa-2 cells and settle there. This type of mutation/selection strategy may be applicable to other combinations of cancer cells and bacterial species.

Girdin regulates the proliferation and apoptosis of pancreatic cancer cells via the PI3K/Akt signalling pathway.

Girdin functions as an Akt phosphorylation enhancer (APE), which expedites the proliferation and survival of many types of tumours. However, the influence of Girdin on pancreatic cancer and the underlying molecular mechanisms have yet to be uncovered. Hence, in the present study, we sought to elucidate the function of Girdin in pancreatic cancer malignancy, particularly its role in pancreatic cancer cell proliferation, migration and apoptosis. Immunohistochemistry (IHC) was used to evaluate Girdin expression in pancreatic cancer tissues and to analyse its correlation with pathological grade. Girdin expression was further validated in pancreatic cancer cell lines (AsPC-1, BxPC-3 and PANC-1), and human pancreatic ductal epithelial (HPNE) cells were used as a control. Recombinant adenovirus vectors containing Girdin-siRNA were constructed to inhibit Girdin expression and were used in subsequent experiments to determine the effects of Girdin silencing on pancreatic cancer cells. Girdin silencing suppressed pancreatic cancer cell proliferation and induced pancreatic cancer cell apoptosis in vitro and in vivo. According to the results of further mechanistic investigations, Girdin may regulate cell processes through the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) signalling pathway to exert additive effects on pancreatic cancer.

Chronic ethanol exposure of human pancreatic normal ductal epithelial cells induces cancer stem cell phenotype through SATB2.

The incidence of pancreatic cancer is on the rise. Risk factors for pancreatic cancer include alcohol toxicity and metabolic conditions such as obesity, hypertension, dyslipidaemia, insulin resistance and type 2 diabetes. However, the molecular mechanism by which chronic alcohol consumption contributes to pancreatic cancer is not well understood. The purpose of the study was to demonstrate the effects of long‐term chronic ethanol exposure on the transformation of human pancreatic normal ductal epithelial (HPNE) cells. Our data showed that ethanol‐transformed HPNE cells were more progressively transformed exhibiting spheroids and colonies, and anchorage‐independent growth. These transformed cells contained high levels of reactive oxygen species and induced SATB2 expression. Furthermore, during ethanol‐induced cellular transformation, cells gained the phenotypes of cancer stem cells (CSCs) by expressing pluripotency maintaining factors (Oct4, Sox2, cMyc and KLF4) and stem cell markers (CD24, CD44 and CD133). Ethanol‐induced SATB2 can bind to the promoters of KLF4, Oct4, cMyc, Sox2, Bcl‐2 and XIAP genes. Suppression of SATB2 expression in ethanol‐transformed HPNE cells inhibited cell proliferation, colony formation and markers of CSCs and pluripotency. These data suggest that chronic alcohol consumption may contribute toward the development of pancreatic cancer by converting HPNE cells to cancer stem‐like cells.

Antimicrobial Peptide Human Neutrophil Peptide 1 as a Potential Link Between Chronic Inflammation and Ductal Adenocarcinoma of the Pancreas.

Defensins are antimicrobial peptides playing a role in innate immunity, in epithelial cell regeneration, and in carcinogenesis of inflammation-triggered malignancies. We analyzed this role in pancreatic ductal adenocarcinoma (PDAC) in the context of its association with chronic pancreatitis (CP). Human tissue of healthy pancreas, CP, and PDAC was screened for defensins by immunohistochemistry. Defensin α 1 (human neutrophil peptide 1 [HNP-1]) expression was validated using mass spectrometry and microarray analysis. Human neutrophil peptide 1 expression and influences of proinflammatory cytokines (tumor necrosis factor α, interleukin 1β, and interferon γ) were studied in human pancreatic cancer cells (Colo 357, T3M4, PANC-1) and normal human pancreatic duct epithelial cells (HPDE). Accumulation of HNP-1 in malignant pancreatic ductal epithelia was seen. Spectrometry showed increased expression of HNP-1 in CP and even more in PDAC. At RNA level, no significant regulation was found. In cancer cells, HNP-1 expression was significantly higher than in HPDE. Proinflammatory cytokines significantly led to increased HNP-1 levels in culture supernatants and decreased levels in lysates of cancer cells. In HPDE cytokines significantly decreased HNP-1 levels. Inflammatory regulation of HNP-1 in PDAC tissue and cells indicates that HNP-1 may be a link between chronic inflammation and malignant transformation in the pancreas.

Selective Treatment of Pancreatic Cancer Cells by Plasma-Activated Saline Solutions

Atmospheric discharge between a metal pin cathode and saline solution (SS) anode with different air gap lengths was considered for investigating plasma self-organization patterns at the liquid anode surface. We report the effect of plasma-activated SSs on human pancreas adenocarcinoma cancer cells line (BxPC-3) and human pancreatic duct epithelial normal cells line (H6c7). The presence of reactive oxygen species and reactive nitrogen species resulted in the anti-cancer properties of plasma-activated SS.