BxPC-3 Pancreatic Cancer Cells Research Articles

Salidroside ameliorated hypoxia-induced tumorigenesis of BxPC-3 cells via downregulating hypoxia-inducible factor (HIF)-1α and LOXL2.

Herein, we found that salidroside suppressed hypoxia‐inducible factor 1 alpha (HIF‐1α) and lysyl oxidase‐like protein 2 (LOXL2) within human pancreatic cancer BxPC‐3 cells cultured both under normoxia and hypoxia condition. To investigate the effect of salidroside on tumorigenesis of BxPC‐3 cells and whether HIF‐1α and LXCL2 were involved in this process, cells transfected with or without LOXL2 overexpression vector, were treated with 50 μg/mL of salidroside or 50 μM of KC7F2 (a HIF‐1α inhibitor) under hypoxia. Cell viability and invasion were assessed using CCK‐8 and Transwell chamber assay, respectively. Expression of E‐cadherin and matrix metalloproteinase 2/9 (MMP 2/9) was determined, by Western blot analysis, to assess cell mobility at molecular levels. We confirmed that hypoxia increased LOXL2 and induced tumorigenesis of BxPC‐3 cells, as evidenced by promoted cell proliferation and invasion, enhanced MMP2/9 while reduced E‐cadherin. Interestingly, hypoxia‐induced carcinogenesis was significantly retarded by both salidroside and KC7F2, however, enhanced with LOXL2 overexpression. Besides, salidroside and KC7F2 reduced LOXL2, and reversed the tumorigenesis of BxPC‐3 cells induced by LOXL2 overexpression. Given the inhibitory effect of salidroside on HIF‐1α expression, our data suggested that: (1) LOXL2 was the mechanism, whereby salidroside and KC7F2 showed inhibitory effect on cancer progression of BxPC‐3 cells; (2) salidroside exerted its anticancer effect, most likely, by a HIF‐1α/LOXL2 pathway. In conclusion, salidroside was a novel therapeutic drug in pancreatic cancer, and downregulation of HIF‐1α and LXCL2 was the underlying mechanism.

Sustained co-delivery of gemcitabine and cis-platinum via biodegradable thermo-sensitive hydrogel for synergistic combination therapy of pancreatic cancer

Pancreatic cancer is one of the most devastating cancers with poor prognosis and no significant change in the survival rate over the past decades. Localized targeted drug delivery through interventional endoscopic ultrasonography-guided fine-needle injection (EUS-FNI) is an attractive and minimally invasive strategy for inoperable pancreatic cancer. An injectable in-situ formed long-lasting drug delivery system is a promising alternative for the localized treatment of pancreatic cancer via EUS-FNI. Here, a biodegradable thermo-sensitive copolymer hydrogel for the co-delivery of anticancer agents gemcitabine (GEM) and cis-platinum (DDP) was developed. This hydrogel is a free flowable liquid at room temperature that changes into a semi-solid hydrogel following injection in response to the physiological temperature. Both in vitro and in vivo drug release behaviors indicate sustained drug release of this delivery system. Synergistic cellular proliferation inhibition and desirable apoptosis promotion have been found when pancreatic cancer Bxpc-3 cells were co-cultured with this GEM-DDP/hydrogel system. After a single intratumoral injection, the dual-drug loaded hydrogel formulation exhibited superior anti-tumor efficacy and minimized systemic side effect on pancreatic cancer xenograft mouse model in comparison to the intravenously injected free GEM and DDP combination. In addition, a strong synergistic therapeutic effect of the GEM-DDP/hydrogel system against pancreatic cancer has been found in vitro and in vivo compared to the single-drug loaded hydrogel composites. The obtained findings suggest this developed thermo-sensitive copolymer hydrogel system as a potential universal carrier for the localized targeted delivery of multi-drugs, for use in a variety of inoperable solid tumors.

The ionophore antibiotic gramicidin A inhibits pancreatic cancer stem cells associated with CD47 down-regulation

BackgroundPancreatic cancer stem cells (CSCs), a special population of cells, renew themselves infinitely and resist to various treatment. Gramicidin A (GrA), an ionophore antibiotic derived from microorganism, can form channels across the cell membrane and disrupt cellular ionic homeostasis, leading to cell dysfunction and death. As reported, the ionophore antibiotic salinomycin (Sal) has been proved to kill CSCs effectively. Whether GrA owns the potential as a therapeutic drug for CSCs still remains unknown. This study investigated the effect of GrA on pancreatic CSCs and the mechanism.MethodsTumorsphere formation assay was performed to assess pancreatic CSCs self-renewal potential. In vitro hemolysis assay was determined to test the borderline concentration of GrA. CCK-8 assay was used to detect pancreatic cancer cell proliferation capability. Flow cytometry was performed to detect cell apoptosis and mitochondrial membrane potential. Scanning and transmission electron microscopy was used to observe ultrastructural morphological changes on cell membrane surface and mitochondria, respectively. Western blot analysis was used to determine relative protein expression levels. Immunofluorescence staining was performed to observe CD47 re-distribution.ResultsGrA at 0.05 μM caused tumorspheres disintegration and decrease in number of pancreatic cancer BxPC-3 and MIA PaCa-2 cells. GrA and Sal both inhibited cancer cell proliferation. The IC50 values of GrA and Sal for BxPC-3 cells were 0.025 μM and 0.363 μM; while for MIA PaCa-2 cells were 0.032 μM and 0.163 μM, respectively. Compared on equal concentrations, the efficacy of GrA was stronger than that of Sal. GrA at 0.1 μM or lower did not cause hemolysis. GrA induced ultrastructural changes, such as the decrease of microvilli-like protrusions on cell surface membrane and the swelling of mitochondria. GrA down-regulated the expression levels of CD133, CD44, and CD47; in addition, CD47 re-distribution was observed on cell surface. Moreover, GrA showed synergism with gemcitabine in suppressing cancer cell proliferation.ConclusionsThe study found that GrA was highly active against pancreatic CSCs. It indicates that GrA exerts inhibitory effects against pancreatic CSCs associated with CD47 down-regulation, implying that GrA might play a positive role in modulating the interaction between macrophages and tumor cells.

One-step preparation of reduction-responsive cross-linked gemcitabine prodrug micelles for intracellular drug delivery

A cross-linkable gemcitabine (GEM)-containing reduction-sensitive polymeric micelles based on the copolymer poly(PEG-co-CMA-co-GEM) was successfully fabricated. The copolymer which synthesized by one-step radical copolymerization of poly(ethylene glycol) methacrylate (PEGMA), 7-(2-Methylacryloylethoxy)-4-methylcoumarin (CMA), and 2-((2-hydroxyethyl)disulfanyl)ethyl acrylate (HSEA-GEM), endowed the micelles with “stealth” surface in circulation, photo cross-linkable property, and reduction-sensitivity. The designed micelles were fabricated by self-assembly of the copolymer in aqueous solution followed by UV-light induced cross-linking of coumarin moieties to enhance stability, which would not disassemble even below critical micelle concentration (CMC) or in non-selective solvent (DMSO/H2O 1:1). In vitro drug release curve demonstrated that the intracellular-mimicking reductive microenvironment could accelerated the GEM release of the prodrug micelles. These micelles could be effectively internalized by BxPC-3 pancreatic cancer cells according to confocal laser scanning microscopy (CLSM) detection and flow cytometry analysis. Meanwhile, methyl thiazolyl tetrazolium (MTT) assay demonstrated that the cross-linked prodrug micelles could efficiently inhibit the proliferation of BxPC-3 cells.

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.

Expression of TMEFF2 in Human Pancreatic Cancer Tissue and the Effects of TMEFF2 Knockdown on Cell, Proliferation, and Apoptosis in Human Pancreatic Cell Lines.

BackgroundThe TMEFF2 gene encodes the transmembrane protein with EGF like and two follistatin-like domains 2 and has been reported to be a tumor suppressor gene, but its role remains unknown in pancreatic cancer. This study aimed to investigate the expression of TMEFF2 in human pancreatic cancer tissue and the effects of knockdown of TMEFF2 on cell, proliferation, and apoptosis in human pancreatic cell lines.Material/MethodsThirty-five samples of human pancreatic tissue and adjacent normal pancreatic tissue, and five human pancreatic cancer cell lines, CAPAN1, ASPC1, BXPC3, SW1990, and CFPAC were studied. RNA expression, protein expression, cell proliferation, and apoptosis were studied using real-time polymerase chain reaction (RT-PCR), Western blot, the cell counting kit-8 (CCK-8) assay, and flow cytometry, respectively. A co-immunoprecipitation assay evaluated protein interactions.ResultsTMEFF2 expression was down-regulated in pancreatic cancer tissue compared with normal pancreas. In human pancreatic cancer cell lines, overexpression of TMEFF2 suppressed cell proliferation and enhanced apoptosis, suppressed the expression of p-STAT3, MCL1, VEGF and increased the expression of the tyrosine-specific protein phosphatase, SHP-1. The co-immunoprecipitation assay showed that TMEFF2 interacted with SHP-1. Knockdown of expression of TMEFF2 resulted in the increased expression of p-STAT3, MCL1, and VEGF, increased cell proliferation and decreased cell apoptosis, which were reversed by overexpression of SHP-1.ConclusionsIn pancreatic cancer, TMEFF2 exerted as a tumor suppressor effect by regulating p-STAT3, MCL1, and VEGF via SHP-1.

The effects of microRNA-181b on proliferation and invasion of pancreatic cancer cells

Objective To investigate the effects of microRNA-181b (miR-181b) on cell proliferation and invasion of pancreatic cancer cells. Methods MiR-181b expression levels in 3 pancreatic cancer cell lines PANC-1, BXPC-3, ASPC-1 and normal pancreatic HTERT-HPNE cells were observed by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). PANC-1 cells were transfected with 50 nmol/L miR-181b inhibitor or negative control using Lipofectamine 2000. After transfection, the expression of miR-181b was measured by RT-qPCR. Cell proliferation was tested by methylthiazol tetrazolium (MTT) assay. Cell invasion was detected by transwell. Bioinformatics prediction and luciferase activity tests were to verify phosphate and tension homology deleted on chromsome ten (PTEN) was the targeted gene of miR-181b. The expression levels of PTEN, matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) were measured by RT-qPCR. Results Compared with the HTERT-HPNE cells (1.00±0.06), the expression level of miR-181b in PANC-1 (2.84±0.16), BXPC-3 (1.74±0.18), ASPC-1 (2.21±0.20) was significantly increased. After transfection with 50 nmol/L miR-181b inhibitor, there was no obvious change in PANC-1 cell proliferation (P>0.05), and cell invasive ability was inhibited (P<0.01). Bioinformatics predicted PTEN might be the targeted gene of miR-181b. Luciferase experiments showed that luciferase activity of wild-type PTEN group fell by 39% after transfection of miR-181b mimics, but there was no statistical difference in the mutant group. After transfection with miR-181b inhibitor, PTEN was significantly increased (1.85±0.06 vs. 1.01±0.08, P<0.05), and MMP-2 (0.59±0.02 vs. 1.01±0.04) and MMP-9 (0.66±0.02 vs. 0.99±0.07) were significantly decreased (P<0.05). Conclusion These results demonstrate that miR-181b promotes proliferation and invasion in pancreatic cancer cells, which may be partly related to effecting MMP-2 and MMP-9 expression by PTEN. Therefore, it may be a new target for the biologic therapy for pancreatic cancer. Key words: Pancreatic cancer; MicroRNA-181b; Proliferation; Invasion

Dual Controlled Delivery of Gemcitabine and Cisplatin Using Polymer-Modified Thermosensitive Liposomes for Pancreatic Cancer.

Although combinational anticancer chemotherapies have been proven to improve the life expectancy of patients in the clinic, their full potential is severely limited by the additive toxicities of the drug molecules. Targeted drug delivery systems could alleviate this major limitation by the design of nanocarriers that can cocarry multiple drug molecules in order to augment drug synergism at the site of interest while reducing the systemic side effects. In this study, we report on a thermoresponsive polymer-coated liposome nanocarrier that is capable to cocarry two potent anticancer drugs and release them via a thermally triggered mechanism. A synthetic polymer ([poly(diethylene glycol) methacrylate-co-poly(oligoethylene glycol) methacrylate]-b-poly(2-ethylhexyl) methacrylate) was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization and was used as a thermoresponsive polymer coating shell on thermosensitive liposome carriers. The formulations were tested in vitro against two pancreatic cancer cell lines, MiaPaCa-2 and BxPC-3, and their cytotoxic potency was studied with respect to their targeted release properties as well as their biological interactions with cellular organelles. The polymer-modified liposomes (PMTLs) could cocarry and release Gemcitabine (Gem) and cisplatin (Cis) in a thermally controlled rate and were also found to exhibit specific hydrophobic interactions with the cell membranes above the temperature transition of the formulations. In addition, the nanocarriers were found to induce more than 10-fold improvement of the IC50 of both drugs, either as monotherapies or in combination, in both cell lines tested, in a temperature dependent manner. The proposed formulations constitute a potent nanomedicinal approach for the codelivery of multiple drug molecules and could find potential uses as thermally triggered drug delivery systems for precision medicine and oncology and also as modulators of drug efficacy at the cellular level owing to their unique interactions with the cell membranes.

ProNGF siRNA inhibits cell proliferation and invasion of pancreatic cancer cells and promotes anoikis

BackgroundPrecursor of nerve growth factor (proNGF) was previously considered biologically inactive; however, it has recently been identified as having important roles in the pathology of cancer development. AimThis study aimed to explore the therapeutic effects of proNGF siRNA on the proliferation, invasion, and anoikis of pancreatic cancer cells and determine the functions of proNGF. MethodsPancreatic ductal adenocarcinoma (PDAC) and paired paracancerous tissue samples were collected from 60 patients for evaluation of proNGF expression by immunohistochemistry staining, qPCR, and western blotting. PDAC cell proliferation, migration, apoptosis, and anoikis following proNGF siRNA knockdown were investigated in two pancreatic cancer cell lines, Panc-1 and Bxpc-3, using BrdU incorporation assays, EdU staining, Ki-67 immunofluorescence (IF) staining, wound-healing assays, transwell invasion assays, and EthD-1 IF staining. Autophagy-related proteins were also measured by western blotting. ResultsLevels of proNGF protein were higher in pancreatic cancer tissues and cells lines than those in paracancerous tissues and normal pancreatic duct epithelial cells, respectively. In vitro, ProNGF knockdown by siRNA led to significantly reduced cell proliferation, remarkably inhibited wound-healing, and reduced the number of invaded PDAC cells in migration and transwell assays. Treatment with proNGF siRNA also downregulated ATG5 and Beclin 1 protein levels, increased those of P62, and increased EthD-1 staining in PDAC cells. ConclusionProNGF expression is elevated in PDAC tissues and cell lines, and proNGF siRNA can inhibit cell proliferation, migration, and invasion, and promote anoikis of pancreatic cancer cells, in which decreased proNGF may participate.

Silencing of long noncoding RNA LINC00958 prevents tumor initiation of pancreatic cancer by acting as a sponge of microRNA-330-5p to down-regulate PAX8

Pancreatic cancer (PC) represents a relatively rare but severe malignancy worldwide. Accumulated studies have emphasized the potential of long noncoding RNA (lncRNA) as therapeutic strategies for several human cancers. Thus, we aimed to investigate whether a novel non-coding RNA regulatory circuitry involved in PC. Aberrantly expressed lncRNAs and mRNAs were screened out of microarray database. Following the determination of RNA expression, PANC-1 and BxPC-3 PC cells were adopted, after which the expression of miR-330-5p, PAX8 and LINC00958 were subsequently altered. RNA crosstalk was validated by dual-luciferase reporter gene assay. In order to detect whether LINC00958 could act as ceRNA to competitively sponge miR-330-5p and regulate PAX8, subcellular location of LINC00958 and interaction between LINC00958 and miR-330-5p were measured by FISH and RNA pull down respectively. The epithelial mesenchymal transition (EMT) process, cell invasion, and tumor growth were determined in vitro and in vivo. LINC00958 and PAX8 were up-regulated, while miR-330-5p was down-regulated during PC. LINC00958 mainly expressed in the cytoplasm and LINC00958 competitively sponged miR-330-5p. Upregulated miR-330-5p or downregulated PAX8 inhibited the EMT process as well as the invasion and metastasis ability of the PC cells. Moreover, the results indicated that miR-330-5p negatively targeted PAX8, and LINC00958 ultimately showcasing its ability to bind to miR-330-5p through its interaction with AGO2. Therefore, silencing of LINC00958 may bind to miR-330-5p to inhibit PAX8 in a competitive fashion, thereby preventing the progression of PC.

Cumulus Cells Are Potential Candidates for Cell Therapy.

Cumulus cells (CCs) originate from the membrane granulosa cells and surround oocytes during follicle maturation. CCs produce high levels of hyaluronan that targets CD44, which is a major tumorigenic marker. This study aimed to investigate whether CCs have a role in cell therapy by targeting CD44 in pancreatic cancer cells. CCs were isolated from the oocytes and incubated in a hypoxic environment. BxPC-3 pancreatic cancer cells were treated with CC conditioned media for three days. Conditioned media of CC cells incubated in hypoxic conditions caused a 25% reduction in the viability of BxPC-3 cells. Expression of anti-apoptotic genes was down-regulated, while that of pro-apoptotic genes was upregulated. An increased number of BxPC-3 cells exhibited increased levels of reactive oxygen species and arrested in the synthesis (S) phase of the cell cycle. CCs conditioned medium induced apoptosis of pancreatic cancer cells.

KRas4B-PDE6\u03b4 complex stabilization by small molecules obtained by virtual screening affects Ras signaling in pancreatic cancer

BackgroundThe GTPase KRas4B has been utilized as a principal target in the development of anticancer drugs. PDE6δ transports KRas4B to the plasma membrane, where it is released to activate various signaling pathways required for the initiation and maintenance of cancer. Therefore, identifying new small molecules that prevent activation of this GTPase by stabilizing the KRas4B-PDE6δ molecular complex is a practical strategy to fight against cancer.MethodsThe crystal structure of the KRas4B-PDE6δ heterodimer was employed to locate possible specific binding sites at the protein-protein interface region. Virtual screening of Enamine-database compounds was performed on the located potential binding sites to identify ligands able to simultaneously bind to the KRas4B-PDE6δ heterodimer. A molecular dynamics approach was used to estimate the binding free-energy of the complex. Cell viability and apoptosis were measured by flow cytometry. G-LISA was used to measure Ras inactivation. Western blot was used to measure AKT and ERK activation. MIA PaCa-2 cells implanted subcutaneously into nude mice were treated with D14 or C22 and tumor volumes were recorded.ResultsAccording to the binding affinity estimation, D14 and C22 stabilized the protein-protein interaction in the KRas4B-PDE6δ complex based on in vitro evaluation of the 38 compounds showing antineoplastic activity against pancreatic MIA PaCa-2 cancer cells. In this work, we further investigated the antineoplastic cellular properties of two of them, termed D14 and C22, which reduced the viability in the human pancreatic cancer cells lines MIA PaCa-2, PanC-1 and BxPC-3, but not in the normal pancreatic cell line hTERT-HPNE. Compounds D14 and C22 induced cellular death via apoptosis. D14 and C22 significantly decreased Ras-GTP activity by 33% in MIA PaCa-2 cells. Moreover, D14 decreased AKT phosphorylation by 70% and ERK phosphorylation by 51%, while compound C22 reduced AKT phosphorylation by 60% and ERK phosphorylation by 36%. In addition, compounds C22 and D14 significantly reduced tumor growth by 88.6 and 65.9%, respectively, in a mouse xenograft model.ConclusionsWe identified two promising compounds, D14 and C22, that might be useful as therapeutic drugs for pancreatic ductal adenocarcinoma treatment.

Curcumin attenuates hyperglycemia-driven EGF-induced invasive and migratory abilities of pancreatic cancer via suppression of the ERK and AKT pathways

The relationship between diabetes mellitus and pancreatic cancer is complex. Diabetes has been postulated to be both an independent risk factor and a consequence of pancreatic cancer. Our previous study confirmed that curcumin plays a vital role in inhibiting the epithelial-mesenchymal transition of pancreatic cancer cells. However, whether curcumin attenuates hyperglycemia-induced cancer invasive and migratory abilities and the underlying mechanisms are not yet well understood. As high glucose is able to induce the expression of epidermal growth factor (EGF), which is intimately related with tumor progression, the aim of this study was to evaluate whether curcumin could influence the high glucose-induced EGF/EGFR pathway and the biological activity of pancreatic cancer cells. Human pancreatic cancer BxPC-3 cells were exposed to high glucose or EGF, with or without curcumin, LY294002 (an Akt inhibitor) or PD98059 (an ERK inhibitor). MTT, Transwell invasion and wound healing assays were used to detect the proliferation, invasion and migration potential of cancer cells. The activation of p-EGFR, p-ERK and p-Akt was determined by western blot analysis. The expression levels of uPA and E-cadherin were examined using qRT-PCR and western blot analysis. The results showed that high glucose could not only promote the proliferation, invasion and migration of pancreatic cancer cells, but also induce the expression of EGF and activation of EGFR, ERK and Akt. These effects of high glucose were counter-balanced by curcumin. EGF-induced proliferative, invasive and migratory abilities of BxPC-3 cells were abrogated by curcumin, LY294002 and PD98059. In addition, EGF-modulated activation of EGFR, ERK and Akt, as well as the expression of uPA and E-cadherin were inhibited by curcumin. Taken together, the present study indicates that curcumin suppresses hyperglycemia-driven EGF-induced invasion and migration of pancreatic cancer cells by inhibiting the EGF/EGFR signaling pathway and its downstream signaling molecules including ERK and Akt. Curcumin is a potential anticancer agent for pancreatic cancer.

β2-AR regulates the expression of AKR1B1 in human pancreatic cancer cells and promotes their proliferation via the ERK1/2 pathway.

Psychological stress has been recognized as a well-documented risk factor associated with β2-adrenergic receptor (β2-AR) in the development of pancreatic cancer. Aldo-keto reductase 1 member B1 (AKR1B1) is a potential interacting partner of β2-AR, but the effect of their interaction on pancreatic cancer cells is not known at present. We found a positive correlation between AKR1B1 and β2-AR expression in pancreatic cancer tissue samples, and co-localization of these proteins in the human pancreatic cancer BXPC-3 cell line. Compared to the controls, the CFPAC-1 and PANC-1 pancreatic cancer cells overexpressing β2-AR and AKR1B1 respectively showed significantly higher proliferation rates, which is attributed to higher proportion of cells in the S phase and decreased percentage of early apoptotic cells. Furthermore, overexpression of β2-AR led to a significant increase in the expression of AKR1B1 and phosphorylated extracellular signal-regulated kinase (p-ERK1/2). Overexpression of AKR1B1 significantly decreased β2-AR levels and increased that of p-ERK1/2. Taken together, β2-AR directly interacted with and up-regulated AKR1B1 in pancreatic cancer cells, and promoted their proliferation and inhibited apoptosis via the ERK1/2 pathway. Our findings also highlight the β2-AR-AKR1B1 axis as a potential therapeutic target for pancreatic cancer.

Overexpression of Gremlin�1 by sonic hedgehog signaling promotes pancreatic cancer progression

Sonic hedgehog (SHH) signaling is an important promotor of desmoplasia, a critical feature in pancreatic cancer stromal reactions involving the activation of pancreatic stellate cells (PSCs). Gremlin 1 is widely overexpressed in cancer-associated stromal cells, including activated PSCs. In embryonic development, SHH is a potent regulator of Gremlin 1 through an interaction network. This subtle mechanism in the cancer microenvironment remains to be fully elucidated. The present study investigated the association between Gremlin 1 and SHH, and the effect of Gremlin 1 in pancreatic cancer. The expression of Gremlin 1 in different specimens was measured using immunohistochemistry. The correlations among clinico-pathological features and levels of Gremlin 1 were evaluated. Primary human PSCs and pancreatic cancer cell lines were exposed to SHH, cyclopamine, GLI family zinc finger-1 (Gli-1) small interfering RNA (siRNA), and Gremlin 1 siRNA to examine their associations and effects using an MTT assay, reverse transcription-quantitative polymerase chain reaction analysis, western blot analysis, and migration or invasion assays. The results revealed the overexpression of Gremlin 1 in pancreatic cancer tissues, mainly in the stroma. The levels of Gremlin 1 were significantly correlated with survival rate and pT status. In addition, following activation of the PSCs, the expression levels of Gremlin 1 increased substantially. SHH acts as a potent promoter of the expression of Gremlin 1, and cyclopamine and Gli-1 siRNA modulated this effect. In a screen of pancreatic cancer cell lines, AsPC-1 and BxPC-3 cells expressed high levels of Gremlin 1, but only AsPC-1 cells exhibited a high expression level of SHH. The results of the indirect co-culture experiment suggested that paracrine SHH from the AsPC-1 cells induced the expression of Gremlin 1 in the PSCs. Furthermore, Gremlin 1 siRNA negatively regulated the proliferation and migration of PSCs, and the proliferation, invasion and epithelial-mesenchymal transition of AsPC-1 and BxPC-3 cells. Based on the data from the present study, it was concluded that an abnormal expression level of Gremlin 1 in pancreatic cancer was induced by SHH signaling, and that the overexpression of Gremlin 1 enabled pancreatic cancer progression.

Interferon Gamma Inhibits CXCL8-Induced Proliferation and Migration of Pancreatic Cancer BxPC-3 Cell Line via a RhoGDI2/Rac1/NF-κB Signaling Pathway.

Interferon gamma (IFN-γ) is a dimeric soluble cytokine and the only type II interferon. Accumulated evidence suggests that IFN-γ inhibits tumor progression. This study investigated the effects of IFN-γ on the proliferation and migration of pancreatic cancer (PC) cells and the underlying mechanism. IFN-γ treatment decreased the expression and secretion of CXCL8 in BxPC-3 PC cells, suppressed the proliferation and migration of these cells, and enhanced their apoptosis, as determined by increased levels of cleaved Caspase-8 and Bax together with reduced expression of Bcl-2. These effects were abolished by overexpression of CXCL8. Moreover, IFN-γ treatment downregulated RhoGDI2 expression. Depletion of RhoGDI2 and Rac1 by using small interfering RNAs and inhibition of NF-κB by BMS-345541 (an IκB kinase [IKK] inhibitor) suppressed expression of CXCL8. Our results indicate that IFN-γ inhibits the proliferation and migration of PC cells by suppressing CXCL8 expression via a RhoGDI2/Rac1/NF-κB signaling pathway.

The marine natural product Scalarin inhibits the receptor for advanced glycation end products (RAGE) and autophagy in the PANC-1 and MIA PaCa-2 pancreatic cancer cell lines.

Pancreatic cancer, the fourth leading cause of cancer death in the United States, has a negative prognosis because metastasis occurs before symptoms manifest. Although combination therapies are showing improvements in treatment, the survival rate for pancreatic cancer five years post diagnosis is only 8%, stressing the need for new treatments. The receptor for advanced glycation end products (RAGE) has recently emerged as a chemotherapeutic target in KRAS driven pancreatic cancers both for treatment and in chemoprevention. RAGE appears to be an important regulator of inflammatory, stress and survival pathways that lead to carcinogenesis, resistance to chemotherapy, enhanced proliferation and the high metastatic potential of pancreatic cancer. RAGE expression has been demonstrated in pancreatic cancer tumors but not in adjacent epithelial tissues. Its presence is associated with increased proliferation and metastasis. In an effort to identify novel inhibitors of RAGE among our collection of marine-derived secondary metabolites, a cell-based screening assay utilizing flow cytometry was developed. This effort led to the identification of scalarin as the active compound in a marine sponge identified as Euryspongia cf. rosea. Scalarin is a sesterterpene natural product isolated previously from a different marine sponge. Scalarin reduces the levels of RAGE and inhibits autophagy in the PANC-1 and MIA PaCa-2 pancreatic cancer cell lines. Its IC50 for cytotoxicity ranges between 20 and 30μM in the AsPC-1, PANC-1, MIA PaCa-2 and BxPC-3 pancreatic cancer cell lines. Inhibition of autophagy limits tumor growth and tumorigenesis in pancreatic cancer, making scalarin an interesting compound that may merit further study.

Combination Effects of Hispidin and Gemcitabine via Inhibition of Stemness in Pancreatic Cancer Stem Cells.

Natural products extracted from plants can be potent for developing pharmaceutical products. Hispidin, a polyphenolic compound mainly derived from the medicinal mushroom Phellinus linteus, has been shown to have a therapeutic potential against cancer cells. Pancreatic cancer is one of the most aggressive solid malignancies with high resistance to existing drugs. Cancer stem cells (CSCs) are responsible for chemoresistance. The present study aimed to evaluate the anticancer effects of hispidin on pancreatic CSCs. The cytotoxic effects of hispidin on BxPC-3 and AsPC-1 pancreatic cancer cells and BxPC-3 CD44+ CSCs and the synergistic effects of gemcitabine and hispidin on CSCs were evaluated by a series of in vitro experiments including the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), fluorescence-activated cell sorting, colony forming, Transwell assay, immunocytochemistry, sphere-forming, and western blot assays. Hispidin exerted antitumor effects against both BxPC-3 pancreatic cancer cells and CSCs. Furthermore, it was found that hispidin sensitized pancreatic CSCs to gemcitabine and promoted the therapeutic efficacy of gemcitabine. Hispidin might be a novel chemosensitizer for gemcitabine and a potential synergistic agent for increasing the therapeutic index of gemcitabine as a treatment for pancreatic cancer.

Abstract 2504: Sialylation of EGFR by the glycosyltransferase ST6Gal-I results in receptor activation and resistance to gefitinib mediated apoptosis

Abstract The Epidermal Growth Factor Receptor (EGFR) is one of the most commonly altered receptors in cancer. In the present study, we report that sialylation of EGFR by the tumor-associated sialyltransferase ST6Gal-I increases receptor activation resulting in resistance to the EGFR tyrosine kinase inhibitor gefitinib. ST6Gal-I adds an α2-6 sialic acid (a bulky, negatively charged sugar) to select cell surface receptors, thereby modulating receptor function. Previously our group has shown that ST6Gal-I activity confers hallmark cancer stem cell characteristics such as invasion, chemoresistance, and resistance to microenvironmental stressors such as growth factor deprivation and hypoxia. Kinomics data collected for this study reveal that in OV4 ovarian cancer cells, where ST6Gal-I has been overexpressed using a lentiviral construct, ST6Gal-I overexpressing cells have an increase in overall tyrosine kinase activity in comparison to cells without ST6Gal-I expression, whereas the overall serine/threonine kinase activity is largely unaffected by ST6Gal-I expression. Specifically, the receptor tyrosine kinase, EGFR, is one of the most significantly altered kinases in ST6Gal-I expressing cells, with ST6Gal-I expressing cells having an increase in overall activity in comparison to non-expressing cells. Based on these findings, we further interrogated the role of ST6Gal-I on EGFR function. To this end, we first confirmed the sialylation status of EGFR in cells with either forced ST6Gal-I overexpression or shRNA knockdown. Using OV4 and Skov3 cells, ovarian cancer cells, as well as BxPC3 pancreatic cancer cells, we conclude that not only is EGFR a substrate of ST6Gal-I, but that sialylation of EGFR by ST6Gal-I results in basal activation of the receptor, independent of EGF ligand treatment. Further, upon stimulation with EGF, receptor activation is increased in ST6Gal-I expressing versus non-expressing cells. To determine the functional significance of EGFR sialylation by ST6Gal-I, we treated cells with differential ST6Gal-I expression with gefitinib, an EGFR tyrosine kinase inhibitor widely used as an anti-cancer therapy. These studies revealed that ST6Gal-I expressing cells were protected from gefitinib mediated apoptosis. Finally, given that EGFR activation has been shown to promote epithelial to mesenchymal transition (EMT), we evaluated ST6Gal-I expressing cells treated with EGF for the EMT marker Slug. We report that while ST6Gal-I expressing cells inherently have elevated Slug expression versus cells without ST6Gal-I, treatment with EGF greatly enhances Slug expression in ST6Gal-I expressing versus non-expressing cells. Collectively, these results reveal a novel functional role for sialylated EGFR and suggest that the sialylation of EGFR may facilitate EMT as well as provide cells a mechanism to overcome chemoresistance. Citation Format: Colleen Britain, Andrew Holdbrooks, Susan Bellis. Sialylation of EGFR by the glycosyltransferase ST6Gal-I results in receptor activation and resistance to gefitinib mediated apoptosis [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 2504.

Abstract 816: Antibody-drug conjugate targeting glypican-1 shows potent antitumor effect in pancreatic cancer

Abstract Pancreatic cancer is the most lethal malignancy; thus developing new treatment options is urgently required. We confirmed that enhanced expression of glypican-1 (GPC1) was observed in pancreatic cancer. In this study, we aimed to develop novel antibody-drug conjugate (ADC) targeting GPC1 as a new therapy for pancreatic cancer. By the immunohistochemical analysis, enhanced expression of GPC1 was observed in pancreatic cancer. Among pancreatic cancer cell lines, BxPC3 and T3M4 cells expressed high expression of GPC1 compared to SUIT2 cells. We developed new anti-GPC1 monoclonal antibody (mAb) and selected a clone having highly internalizing activity. The anti-GPC1 mAb was conjugated with cytotoxic agent monomethyl auristatin F (MMAF). Compared with the control ADC, this GPC1-ADC showed potent antitumor effect toward BxPC3 and T3M4, while little activity in SUIT2 cells. In BxPC3 xenograft model, GPC1-ADC had significant and potent tumor growth inhibition in a dose dependent manner. In addition, using patient derived tumor xenograft (PDX) model with GPC1-positive pancreatic cancer, GPC1-ADC also showed significant tumor growth inhibition. In the tumor tissue, GPC1-ADC-mediated G2/M phase cell cycle arrest was detected in GPC1-ADC treated mice compared to control-ADC treated mice. In summary, our newly developed GPC1-ADC showed significant tumor growth inhibition against not only GPC1-positive pancreatic cell line but also patient derived GPC1-positive pancreatic cancer. Our preclinical data demonstrated that targeting GPC1 by ADC is a promising therapy for patients with GPC1-positive pancreatic cancer. Citation Format: Satoshi Serada, Takahiko Nishigaki, Takahito Sugase, Yurina Saito, Tsuyoshi Takahashi, Kosuke Hiramatsu, Minoru Fujimoto, Masaki Mori, Yuichiro Doki, Tetsuji Naka. Antibody-drug conjugate targeting glypican-1 shows potent antitumor effect in pancreatic cancer [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 816.